U.S. patent application number 17/526509 was filed with the patent office on 2022-09-08 for adjuvant compositions.
This patent application is currently assigned to The Regents of the University of Michigan. The applicant listed for this patent is NanoBio Corporation, The Regents of the University of Michigan. Invention is credited to James R. Baker, JR., Susan Ciotti, Douglas M. Smith.
Application Number | 20220282657 17/526509 |
Document ID | / |
Family ID | 1000006351421 |
Filed Date | 2022-09-08 |
United States Patent
Application |
20220282657 |
Kind Code |
A1 |
Baker, JR.; James R. ; et
al. |
September 8, 2022 |
ADJUVANT COMPOSITIONS
Abstract
The present invention provides methods and compositions for the
stimulation of immune responses. In particular, the invention
provides methods and compositions for enhancing an immune response
to one or more antigens. Compositions and methods of the invention
are useful for the treatment and/or prevention of microbial
infections, such as infections caused by bacteria, viruses, fungi,
and parasites, as well as the treatment and/or prevention of cancer
and malignant diseases. Compositions and methods of the invention
include one or more antigens/immunogens together with an adjuvant
formulation comprising an emulsion delivery system in combination
with one or more immunostimulatory compounds (e.g., a compound that
stimulates the innate immune system (e.g., a toll-like receptor
antagonist (e.g., synthetic oligodeoxynucleotides (ODN)))) that
enhance immune responses to the one or more antigens/immunogens
when administered to a subject. Compositions and methods of the
invention find use in, among other things, clinical (e.g.
therapeutic and preventative medicine (e.g., vaccination)) and
research applications.
Inventors: |
Baker, JR.; James R.; (Ann
Arbor, MI) ; Smith; Douglas M.; (Ann Arbor, MI)
; Ciotti; Susan; (Ann Arbor, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of Michigan
NanoBio Corporation |
Ann Arbor
Ann Arbor |
MI
MI |
US
US |
|
|
Assignee: |
The Regents of the University of
Michigan
Ann Arbor
MI
NanoBio Corporation
Ann Arbor
MI
|
Family ID: |
1000006351421 |
Appl. No.: |
17/526509 |
Filed: |
November 15, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16303074 |
Nov 19, 2018 |
|
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PCT/SE2017/050450 |
May 8, 2017 |
|
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17526509 |
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62338830 |
May 19, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
F01N 3/2892
20130101 |
International
Class: |
F01N 3/28 20060101
F01N003/28 |
Claims
1. An adjuvant composition for modulating an immune response to an
antigen administered to a host, the composition comprising: (a) a
nanoemulsion adjuvant; and (b) at least one immunostimulatory
compound.
2. The adjuvant composition of claim 1, wherein the
immunostimulatory compound is a compound that stimulates the innate
immune system.
3. The adjuvant composition of claim 2, wherein the
immunostimulatory compound is a toll-like receptor antagonist.
4. The adjuvant composition of claim 1, wherein the
immunostimulatory compound is a synthetic oligodeoxynucleotide
(ODN).
5. The adjuvant composition of claim 4, wherein the synthetic ODN
contains a CpG motif.
6. The adjuvant composition of claim 1, wherein the
immunostimulatory compound is a saponin.
7. The adjuvant composition of claim 6, wherein the saponin is a
triterpene glycoside saponin.
8. The adjuvant composition of claim 7, wherein the triterpene
glycoside saponin is QS-21.
9. An immunogenic composition for eliciting an immune response in a
subject comprising: (a) one or more antigens; (b) a nanoemulsion
adjuvant; and (c) at least one immunostimulatory compound.
10. The immunogenic composition of claim 9, wherein the one or more
antigens are selected from an inactivated microbial pathogen, an
isolated and/or recombinant peptide, an isolated and/or recombinant
protein, a glycoprotein, a lipoprotein, a glycopeptide, a
lipopeptide, a toxoid, a carbohydrate, and a tumor-specific
antigen.
11. The immunogenic composition of claim 9, wherein the
nanoemulsion comprises oil, water, an organic solvent, a cationic
surfactant, and an nonionic surfactant.
12. The immunogenic composition of claim 9, wherein the
immunostimulatory compound is a toll-like receptor antagonist.
13. The immunogenic composition of claim 9, wherein the
immunostimulatory compound is a synthetic oligodeoxynucleotide
(ODN).
14. The immunogenic composition of claim 13, wherein the synthetic
ODN contains a CpG motif.
15. The immunogenic composition of claim 9, wherein the
immunostimulatory compound is a saponin.
16. The immunogenic composition of claim 15, wherein the saponin is
a triterpene glycoside saponin.
17. The adjuvant composition of claim 16, wherein the triterpene
glycoside saponin is QS-21.
18. A method of generating an immune response in a subject
comprising administering to the subject an immunogenic composition
comprising: (a) one or more antigens; (b) a nanoemulsion adjuvant;
and (c) at least one immunostimulatory compound.
19. The method of claim 18, wherein the immune response is greater
than the immune response generated in a subject administered a
composition containing the one or more antigens and only the
nanoemulsion adjuvant, and/or is greater than the immune response
generated in a subject administered a composition containing the
one or more antigens and only the immunostimulatory compound.
20. The method of claim 19, wherein the immune response is
synergistically greater.
Description
FIELD OF THE INVENTION
[0001] The present invention provides methods and compositions for
the stimulation of immune responses. In particular, the invention
provides methods and compositions for enhancing an immune response
to one or more antigens. Compositions and methods of the invention
are useful for the treatment and/or prevention of microbial
infections, such as infections caused by bacteria, viruses, fungi
and parasites. Compositions and methods of the invention include
one or more antigens/immunogens together with an adjuvant
formulation comprising an emulsion delivery system in combination
with one or more immunostimulatory compounds (e.g., a compound that
stimulates the innate immune system (e.g., a toll-like receptor
antagonist (e.g., synthetic oligodeoxynucleotides (ODN)))) that
enhance immune responses to the one or more antigens/immunogens
when administered to a subject. Compositions and methods of the
invention find use in, among other things, clinical (e.g.
therapeutic and preventative medicine (e.g., vaccination)) and
research applications.
BACKGROUND
[0002] The body's immune system activates a variety of mechanisms
for attacking pathogens (See, e.g., Janeway, Jr, C A. and Travers
P., eds., in Immunobiology, "The Immune System in Health and
Disease," Second Edition, Current Biology Ltd., London, Great
Britain (1996)). However, not all of these mechanisms are
necessarily activated after immunization. Protective immunity
induced by immunization is dependent upon the capacity of an
immunogenic composition to elicit the appropriate immune response
to resist or eliminate the pathogen. Depending on the pathogen,
cell-mediated and/or humoral immune responses are important for
pathogen neutralization and/or elimination.
[0003] Many antigens are poorly immunogenic or non-immunogenic when
administered by themselves. Strong adaptive immune responses to
antigens generally require that the antigens be administered
together with an adjuvant, a substance that enhances the immune
response (See, e.g., Audbert, F. M. and Lise. L. D. 1993 Immunology
Today, 14: 281-284).
[0004] Killed or subunit vaccines are often poorly immunogenic, and
can result in weak and transient T-cell responses, thus requiring
adjuvants to boost the immune response. However, many currently
available vaccines include adjuvants that are suboptimal with
respect to the quality and magnitude of immune responses they
induce. For example, alum, the only approved adjuvant for use in
humans in the United States, induces relatively good Th2 type
immune responses but is not a potent adjuvant for Th1-type immune
responses (See, e.g., HogenEsch et al., Vaccine (2002) 20 Suppl
3:S34-39). Thus, there is a need for additional effective and safer
adjuvants.
[0005] Two broad categories of adjuvants exist-adjuvant delivery
systems and immunostimulatory compounds/adjuvants. Delivery systems
include particulate formulations such as microparticles, liposomes
and emulsions. The mechanism of action of these systems are not
fully understood but are thought to involve increased uptake by
antigen presenting cells (APC) and/or formation of a depot at the
site of injection. Immunostimulatory adjuvants stimulate innate
immunity resulting in the secretion of cytokines and upregulation
of costimulatory molecules. These events are now known to play an
instructional role in the development of adaptive immune
responses.
[0006] A major impediment in the development of novel vaccines has
been the lack of safe yet effective vaccine adjuvants. In recent
years, synthetic oligodeoxynucleotides containing CpG motifs (CpG)
have gained considerable interest as vaccine adjuvants owing to
their inherent ability to induce and enhance Th1-type immunity.
Through their direct interaction with Toll-like receptor 9 (TLR9)
on human B cells and dendritic cells (DC), as well as indirect
effects on other immune cells such as monocytes, macrophages, and T
cells, CpG enhance antigen presentation and induce the production
of high levels of Th1 cytokines, resulting in the production of
potent antigen-specific Th1-type immune responses. CpG have been
shown to enhance both humoral and cellular immunity in multiple
species including humans. QS-21 is a triterpene glycoside "saponin"
isolated from the bark of the Quillaja saponaria Molina tree, a
species native to South America. The bark of this tree,
particularly the saponin fraction present in the bark, has long
been known as a source of immune stimulators that can be used as
vaccine adjuvants. Espinet (1951) noted the adjuvant activity of
plant saponins to enhance the potency of foot-and-mouth disease
vaccines. Unlike most other immunostimulators, QS-21 is
water-soluble and has been shown to stimulate both humoral and
cell-mediated Th1 and CTL responses to subunit antigens. (See
Dalsgaard 1974).
SUMMARY OF THE INVENTION
[0007] The present invention provides methods and compositions for
the stimulation of immune responses. In particular, the invention
provides methods and compositions for enhancing an immune response
to one or more antigens. Compositions and methods of the invention
are useful for the treatment and/or prevention of microbial
infections, such as infections caused by bacteria, viruses, fungi
and parasites. Compositions and methods of the invention include
one or more antigens/immunogens together with an adjuvant
formulation comprising an emulsion delivery system in combination
with one or more immunostimulatory compounds (e.g., a compound that
stimulates the innate immune system (e.g., a toll-like receptor
antagonist (e.g., synthetic oligodeoxynucleotides (ODN)))) that
enhance immune responses to the one or more antigens/immunogens
when administered to a subject. Compositions and methods of the
invention find use in, among other things, clinical (e.g.
therapeutic and preventative medicine (e.g., vaccination)) and
research applications.
[0008] Accordingly, the present invention provides improved
adjuvant compositions. In one embodiment, the invention provides an
adjuvant formulation for modulating an immune response (e.g.,
enhancing an immune response (e.g., synergistically enhancing an
immune response) to an antigen administered to a subject, the
composition comprising: (a) a nanoemulsion adjuvant; and (b) at
least one immunostimulatory compound (e.g., a compound that
stimulates the innate immune system (e.g., a toll-like receptor
antagonist (e.g., synthetic oligodeoxynucleotides (ODN))). The
adjuvant formulation provided herein exhibit a surprisingly
unexpected adjuvanting effect on an antigen which is greater than
the adjuvanting effect attainable by one of the constituents of the
adjuvant formulation alone. For example, in one embodiment, an
immune response induced in a subject when an immunogenic
compositions of the invention comprising one or more
antigens/immunogens together with an adjuvant formulation
comprising an emulsion in combination with one or more
immunostimulatory compounds is administered to a subject may be
additive or synergistic (e.g., synergistic compared to when the one
or more antigens/immunogens are administered with only the emulsion
(e.g., a nanoemulsion described herein); or when the one or more
antigens/immunogens are administered with only the
immunostimulatory compound (e.g., a compound that stimulates the
innate immune system (e.g., a toll-like receptor antagonist (e.g.,
synthetic oligodeoxynucleotides (ODN)))).
[0009] The adjuvant formulation is not limited by the type of
nanoemulsion utilized. Indeed, a variety of nanoemulsions find use
in an adjuvant formulation including, but not limited to, those
nanoemulsions described herein. Similarly, the present invention is
not limited to any particular immunostimulatory compound. Indeed,
any immunostimulatory compound described herein finds use in an
adjuvant formulation (e.g., for use in an immunogenic composition)
of the invention. In a preferred embodiment, the immunostimulatory
compound is a compound that stimulates the innate immune system
(e.g., a toll-like receptor antagonist (TLR) (e.g., synthetic
oligodeoxynucleotides (ODN)))). However, the invention is not
limited to these immunostimulatory compounds. For example, in some
embodiments, an immunostimulatory compound (e.g., for use in
combination with a nanoemulsion in an adjuvant formulation of the
invention) includes, but is not limited to, a TLR antagonist (e.g.,
a polyinosinic-polycytidylic acid (poly (IC)) (e.g., that activates
TLR3); Pam3CSK4 (e.g., that activates TLR1/2); FSL-1 and/or MALP2
(e.g., that activate TLR2/6); monophosphoryl lipid A, (MPL) (e.g.,
that activate TLR4); flagellin (e.g., that activate TLR5);
imiquimod (e.g., that activate TLR7): a Class B CpG
phosphorothioate oligodeoxynucleotide, (CpG ODN) (e.g., that
activate TLR9); a multi-pattern recognition receptors (multi-PRR
ligand) (e.g., that act as agonists for dual or multiple TLR
receptors and other PRRs)); a RIG-I-like receptor agonist (e.g.,
5'ppp dsRNA and/or Poly(I:C)/LyoVec complexes); NOD and NOD-like
receptor ligand agonists; inflammasome inducers (e.g., alum, alum
salts, and/or alum crystals, chitosan, etc.): cytosolic DNA Sensors
(CDS) and STING Ligands: immune cell receptors (e.g., CD40L,
FMS-like tyrosine kinase ligand (e.g., Flt3 ligand), and multimeric
immune cell receptors); Cytokines and chemokines (e.g., IL-1, IL-6,
TGF-.beta., IL-12, IL-15, interferons (.alpha., .beta., .gamma.,
.delta.), GM-CSF and others); synthetic glycolipids (e.g.,
alpha-galactosylceramide); and/or vitamins (e.g., retinoic
acid).
[0010] In another embodiment, the invention provides an immunogenic
composition for eliciting an immune response in a subject,
including a human, the composition comprising: (a) one or more
antigens; (b) a nanoemulsion; and (c) at least one
immunostimulatory compound.
[0011] In another embodiment, the invention provides a method of
modulating an immune response (e.g., enhancing an immune response)
to an antigen comprising combining the antigen with the adjuvant
formulation of the present invention and administering the same to
a subject (e.g., administering an effective amount to generate a
desired immune response in a subject). The invention is not limited
by the type of antigen combined with an adjuvant formulation of the
invention. Antigens include, but are not limited to, an inactivated
microbial pathogen, an isolated and/or recombinant peptide, an
isolated and/or recombinant protein, a glycoprotein, a lipoprotein,
a glycopeptide, a lipopeptide, a toxoid, a carbohydrate, and/or a
tumor-specific antigen. In some embodiments, an immunogenic
composition of the invention is formulated to comprise between 0.1
and 500 .mu.g of a protein antigen (e.g., derived or isolated from
a pathogen and/or a recombinant form of an immunogenic pathogen
component). However, the present invention is not limited to this
amount of protein antigen (e.g., in some embodiments, more than 500
.mu.g of protein antigen is used, and in other embodiments, less
than 0.1 .mu.g of protein antigen is used). Mixtures of two or more
antigens may be employed. In a preferred embodiment, the peptides,
glycopeptides or lipopeptides include an amino acid sequence
corresponding to an antigenic determinant of a microbial pathogen
that is known in the art. In one embodiment, the protein is
influenza hemagglutinin.
[0012] In another embodiment, the invention provides a method of
generating an immune response in a subject, including a human,
comprising administering thereto an immunogenic composition of the
present invention (e.g., comprising one or more antigens/immunogens
together with an adjuvant formulation comprising an emulsion in
combination with one or more immunostimulatory compounds). The
invention is not limited by the type of immune response generated
and refers to any type of immune response, including, but not
limited to, innate immune responses (e.g., activation of Toll
receptor signaling cascade), cell-mediated immune responses (e.g.,
responses mediated by T cells (e.g., antigen-specific T cells) and
non-specific cells of the immune system), and humoral immune
responses (e.g., responses mediated by B cells (e.g., via
generation and secretion of antibodies into the plasma, lymph,
and/or tissue fluids). The term "immune response" is meant to
encompass all aspects of the capability of a subject's immune
system to respond to antigens and/or immunogens (e.g., both the
initial response to an immunogen (e.g., a pathogen) as well as
acquired (e.g., memory) responses that are a result of an adaptive
immune response). Immune responses include, but are not limited to,
detectable alteration (e.g., increase) in Toll-like receptor (TLR)
activation, lymphokine (e.g., cytokine (e.g., Th1 or Th2 type
cytokines) or chemokine) expression and/or secretion, macrophage
activation, dendritic cell activation, T cell activation (e.g.,
CD4+ or CD8+ T cells), NK cell activation, B cell activation (e.g.,
antibody generation and/or secretion), binding of an immunogen
(e.g., antigen (e.g., immunogenic polypeptide)) to an MHC molecule
and inducing a cytotoxic T lymphocyte ("CTL") response, inducing a
B cell response (e.g., antibody production), T-helper lymphocyte
response, a delayed type hypersensitivity (DTH) response against
antigen (e.g., from which an immunogenic polypeptide is derived),
expansion (e.g., growth of a population of cells) of cells of the
immune system (e.g., T cells, B cells (e.g., of any stage of
development (e.g., plasma cells), and/or increased processing and
presentation of antigen by antigen presenting cells.
[0013] In another embodiment, the invention provides a kit for
preparing an immunogenic composition, comprising: (a) means for
containing a nanoemulsion; (b) means for containing one or more
immunostimulatory compounds; (c) means for containing at least one
antigen; and (d) means for combining the nanoemulsion, one or more
immunostimulatory compounds and at least one antigen to produce the
immunogenic composition.
BRIEF DESCRIPTION OF THE FIGURES
[0014] The following figures form part of the present specification
and are included to further demonstrate certain aspects and
embodiments of the present invention. The invention may be better
understood by reference to one or more of these figures in
combination with the description of specific embodiments presented
herein.
[0015] FIG. 1 shows the vaccination timeline for evaluation of
nanoemulsion adjuvant in combination with QS21 or CpG ODN or using
nanoemulsion adjuvants formulated with DODAC cationic surfactant by
the intramuscular route in CD-1 mice in one embodiment of the
invention.
[0016] FIG. 2 shows endpoint rH5-specific IgG titers upon
intramuscular administration using adjuvant formulations detailed
in Table 8. Serum rH5-specific IgG was determined by ELISA at week
4 (2 weeks post-second immunization).
[0017] FIG. 3 shows cytokine release profiles for I.M. nanoemulsion
dose range experiments: Comparison of the 5% DODAC (single cationic
surfactant) vs. 5% DODAC+CPC (dual cationic surfactant) adjuvant
formulations.
[0018] FIG. 4 shows evaluation of serum rH5-specific antibody
responses after intramuscular immunization of CD-1 mice using
nanoemulsion formulations containing CPC or DODAC (DD).
[0019] FIG. 5 shows serum rH5-specific antibody responses after
intramuscular immunization of CD-1 mice using W805EC alone or in
combination with CpG oligodeoxynucleotide (ODN) 1826. (A) 5% W805EC
in combination with 20 .mu.g CpG ODN and 10 .mu.g rH5 antigen
stimulated high levels (GMT>107) of rH5-specific IgG in serum
after 3 immunizations (Week 6). (B) The combination of W805EC+CpG
also activated the highest levels of "functional" HAI antibody
(GMT: 160; 75% responders) when compared to W805EC alone (GMT:
32:50% responders), or CpG alone (GMT: 77; 50% responders).
[0020] FIG. 6 shows the profile of cell-mediated immune responses
measured by cytokine release upon rH5 antigen re-stimulation of
spleen cells obtained after intramuscular immunization using
nanoemulsion+CpG ODN combination vaccines.
[0021] FIG. 7 shows serum rH5-specific antibody responses after
intramuscular immunization of CD-1 mice using W805EC alone or in
combination with QS21.
[0022] FIG. 8 shows the profile of cell-mediated immune responses
stimulated by nanoemulsion+QS21 combination vaccines when
administered by the intramuscular route in CD-1 mice.
[0023] FIG. 9 show the experimental timeline for evaluation of
DODAC+CpG ODN nanoemulsion combination adjuvant in CD-1 mice.
Buffer system for vaccine formulation: PBS; rH5 (10 .mu.g/animal);
8 animals per group/64 animals; Three IM immunizations each
delivered in a volume of 50 .mu.l at 0, 2, and 4 wks; Sacrifice at
week 6.
[0024] FIG. 10 shows the particle size distribution for NE+CpG ODN
combination vaccines after storage at 5.degree. C.
[0025] FIG. 11 shows the particle size distribution for NE+CpG ODN
combination vaccines after storage at 5.degree. C.
[0026] FIG. 12 shows the particle size distribution for NE+CpG ODN
combination vaccines after storage at 5.degree. C.
[0027] FIG. 13 shows the evaluation of rH5 antigen concentration by
SRID analysis for NE+CpG ODN combination vaccines after storage at
5.degree. C. The concentration of rH5 was calculated after
measurement of well-diameters.
DEFINITIONS
[0028] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below.
[0029] As used herein, the term "microorganism" refers to any
species or type of microorganism, including but not limited to,
bacteria, viruses, archaea, fungi, protozoans, mycoplasma, prions,
and parasitic organisms. The term microorganism encompasses both
those organisms that are in and of themselves pathogenic to another
organism (e.g., animals, including humans, and plants) and those
organisms that produce agents that are pathogenic to another
organism, while the organism itself is not directly pathogenic or
infective to the other organism.
[0030] As used herein the term "pathogen," and grammatical
equivalents, refers to an organism (e.g., biological agent),
including microorganisms, that causes a disease state (e.g.,
infection, pathologic condition, disease, etc.) in another organism
(e.g., animals and plants) by directly infecting the other
organism, or by producing agents that causes disease in another
organism (e.g., bacteria that produce pathogenic toxins and the
like). "Pathogens" include, but are not limited to, viruses,
bacteria, archaea, fungi, protozoans, mycoplasma, prions, and
parasitic organisms.
[0031] The terms "bacteria" and "bacterium" refer to all
prokaryotic organisms, including those within all of the phyla in
the Kingdom Procaryotae. It is intended that the term encompass all
microorganisms considered to be bacteria including Mycoplasma,
Chlamydia. Actinomyces, Streptomyces, and Rickettsia. All forms of
bacteria are included within this definition including cocci,
bacilli, spirochetes, spheroplasts, protoplasts, etc.
[0032] As used herein, the term "fungi" is used in reference to
eukaryotic organisms such as molds and yeasts, including dimorphic
fungi.
[0033] As used herein the terms "disease" and "pathologic
condition" are used interchangeably, unless indicated otherwise
herein, to describe a deviation from the condition regarded as
normal or average for members of a species or group (e.g., humans),
and which is detrimental to an affected individual under conditions
that are not inimical to the majority of individuals of that
species or group. Such a deviation can manifest as a state, signs,
and/or symptoms (e.g., diarrhea, nausea, fever, pain, blisters,
boils, rash, hyper-immune responses, hyper-sensitivity, immune
suppression, inflammation, etc.) that are associated with any
impairment of the normal state of a subject or of any of its organs
or tissues that interrupts or modifies the performance of normal
functions. A disease or pathological condition may be caused by or
result from contact with a microorganism (e.g., a pathogen or other
infective agent (e.g., a virus or bacteria)), may be responsive to
environmental factors (e.g., allergens, malnutrition, industrial
hazards, and/or climate), may be responsive to an inherent defect
of the organism (e.g., genetic anomalies) or to combinations of
these and other factors.
[0034] The terms "host" or "subject," as used herein, refer to an
individual to be treated by (e.g., administered (e.g., injectably
administered)) compositions and methods of the present invention.
Subjects include, but are not limited to, mammals (e.g., murines,
simians, equines, bovines, porcines, canines, felines, and the
like), and most preferably includes humans. In the context of the
invention, the term "subject" generally refers to an individual who
will be administered (e.g., injectably administered) or who has
been administered one or more compositions of the present invention
(e.g., an injectable composition for inducing an immune response
comprising a select nanoemulsion formulated for injection and one
or more antigens).
[0035] As used herein, the terms "inactivating," "inactivation" and
grammatical equivalents, when used in reference to a microorganism
(e.g., a pathogen (e.g., a bacterium or a virus)), refer to the
killing, elimination, neutralization and/or reducing of the
capacity of the microorganism (e.g., a pathogen (e.g., a bacterium
or a virus)) to infect and/or cause a pathological response and/or
disease in a host.
[0036] As used herein, the term "fusigenic" is intended to refer to
an emulsion that is capable of fusing with the membrane of a
microbial agent (e.g., a bacterium or bacterial spore). Specific
examples of fusigenic emulsions are described herein.
[0037] As used herein, the term "lysogenic" refers to an emulsion
(e.g., a nanoemulsion) that is capable of disrupting the membrane
of a microbial agent (e.g., a virus (e.g., viral envelope) or a
bacterium or bacterial spore). In preferred embodiments of the
present invention, the presence of a lysogenic and a fusigenic
agent in the same composition produces an enhanced inactivating
effect compared to either agent alone. Methods and compositions
(e.g., for inducing an immune response (e.g., used as a vaccine)
using this improved antimicrobial composition are described in
detail herein.
[0038] The term "emulsion," as used herein, includes classic
oil-in-water or water in oil dispersions or droplets, as well as
other lipid structures that can form as a result of hydrophobic
forces that drive apolar residues (e.g., long hydrocarbon chains)
away from water and drive polar head groups toward water, when a
water immiscible oily phase is mixed with an aqueous phase. These
other lipid structures include, but are not limited to,
unilamellar, paucilamellar, and multilamellar lipid vesicles,
micelles, and lamellar phases. Similarly, the term "nanoemulsion,"
as used herein, refers to oil-in-water dispersions comprising small
lipid structures. For example, in some embodiments, the
nanoemulsions comprise an oil phase having droplets with a mean
particle size of approximately 0.1 to 5 microns (e.g., about 100,
150, 200, 250, 300, 350, 400, 450, 500 nm or larger in diameter),
although smaller and larger particle sizes are contemplated. The
terms "emulsion" and "nanoemulsion" and "NE" are often used herein,
interchangeably, to refer to the nanoemulsions of the present
invention.
[0039] As used herein, the terms "contact," "contacted," "expose,"
and "exposed," when used in reference to a nanoemulsion and a live
microorganism, refer to bringing one or more nanoemulsions into
contact with a microorganism (e.g., a pathogen) such that the
nanoemulsion inactivates the microorganism or pathogenic agent, if
present. The present invention is not limited by the amount or type
of nanoemulsion used for microorganism inactivation. A variety of
nanoemulsion that find use in the present invention are described
herein and elsewhere (e.g., nanoemulsions described in U.S. Pat.
Apps. 20020045667 and 20040043041, and U.S. Pat. Nos. 6,015,832,
6,506,803, 6,635,676, and 6,559,189, each of which is incorporated
herein by reference in its entirety for all purposes). Ratios and
amounts of nanoemulsion (e.g., sufficient for inactivating the
microorganism (e.g., virus inactivation)) and microorganisms (e.g.,
sufficient to provide an antigenic composition (e.g., a composition
capable of inducing an immune response)) are contemplated in the
present invention including, but not limited to, those described
herein.
[0040] The term "surfactant" refers to any molecule having both a
polar head group, which energetically prefers solvation by water,
and a hydrophobic tail that is not well solvated by water. The term
"cationic surfactant" refers to a surfactant with a cationic head
group. The term "cationic lipid" refers to a positively charged
molecule comprising a positively charged head group and one or more
(e.g., two, three or more) hydrocarbon chains. The term "anionic
surfactant" refers to a surfactant with an anionic head group. When
a single surfactant molecule exhibit both anionic and cationic
dissociations it is called amphoteric or zwitterionic. For example,
this is the case of synthetic products like betaines or
sulfobetaines and natural substances such as aminoacids and
phospholipids. Polymeric surfactants and surface active polymers
result from the association of one or several macromolecular
structures exhibiting hydrophilic and lipophilic characters, either
as separated blocks or as grafts.
[0041] The terms "Hydrophile-Lipophile Balance Index Number" and
"HLB Index Number" refer to an index for correlating the chemical
structure of surfactant molecules with their surface activity. The
HLB Index Number may be calculated by a variety of empirical
formulas as described, for example, by Meyers, (See, e.g., Meyers,
Surfactant Science and Technology, VCH Publishers Inc., New York,
pp. 231-245 (1992)), incorporated herein by reference. As used
herein where appropriate, the HLB Index Number of a surfactant is
the HLB Index Number assigned to that surfactant in McCutcheon's
Volume 1: Emulsifiers and Detergents North American Edition, 1996
(incorporated herein by reference). The HLB Index Number ranges
from 0 to about 70 or more for commercial surfactants. Hydrophilic
surfactants with high solubility in water and solubilizing
properties are at the high end of the scale, while surfactants with
low solubility in water that are good solubilizers of water in oils
are at the low end of the scale.
[0042] As used herein the term "interaction enhancers" refers to
compounds that act to enhance the interaction of an emulsion with a
microorganism (e.g., with a cell wall of a bacteria (e.g., a Gram
negative bacteria) or with a viral envelope (e.g., Vaccinia virus
envelope)). Contemplated interaction enhancers include, but are not
limited to, chelating agents (e.g., ethylenediaminetetraacetic acid
(EDTA), ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), and
the like) and certain biological agents (e.g., bovine serum albumin
(BSA) and the like).
[0043] The terms "buffer" or "buffering agents" refer to materials,
that when added to a solution, cause the solution to resist changes
in pH.
[0044] The terms "reducing agent" and "electron donor" refer to a
material that donates electrons to a second material to reduce the
oxidation state of one or more of the second material's atoms.
[0045] The term "monovalent salt" refers to any salt in which the
metal (e.g., Na, K. or Li) has a net 1+ charge in solution (i.e.,
one more proton than electron).
[0046] The term "divalent salt" refers to any salt in which a metal
(e.g., Mg, Ca, or Sr) has a net 2+ charge in solution.
[0047] The terms "chelator" or "chelating agent" refer to any
materials having more than one atom with a lone pair of electrons
that are available to bond to a metal ion.
[0048] The term "solution" refers to an aqueous or non-aqueous
mixture.
[0049] As used herein, the term "a composition for inducing an
immune response" refers to a composition that, once administered to
a subject (e.g., once, twice, three times or more (e.g., separated
by weeks, months or years)), stimulates, generates and/or elicits
an immune response in the subject (e.g., resulting in total or
partial immunity to a microorganism (e.g., pathogen) capable of
causing disease). In preferred embodiments of the invention, the
composition comprises one or more antigens/immunogens together with
an adjuvant formulation comprising an emulsion delivery system in
combination with one or more immunostimulatory compounds (e.g., a
compound that stimulates the innate immune system (e.g., a
toll-like receptor antagonist (e.g., synthetic
oligodeoxynucleotides (ODN) (e.g., ODN containing immunostimulatory
CpG motifs (CpG))) or a saponin (e.g., a triterpene glycoside
saponin (e.g., QS-21))) formulated for administration (e.g., via
injectable route (e.g., intradermal, intramuscular, subcutaneously,
etc.), mucosal route (e.g., nasally or vaginally), or other route)
to a subject. In further preferred embodiments, the immunogenic
composition comprises one or more other compounds or agents
including, but not limited to, therapeutic agents, physiologically
tolerable liquids, gels, carriers, diluents, adjuvants, excipients,
salicylates, steroids, immunosuppressants, immunostimulants,
antibodies, cytokines, antibiotics, binders, fillers,
preservatives, stabilizing agents, emulsifiers, and/or buffers. An
immune response may be an innate (e.g., a non-specific) immune
response or a learned (e.g., acquired) immune response (e.g. that
decreases the infectivity, morbidity, or onset of mortality in a
subject (e.g., caused by exposure to a pathogenic microorganism) or
that prevents infectivity, morbidity, or onset of mortality in a
subject (e.g., caused by exposure to a pathogenic microorganism)).
Thus, in some preferred embodiments, an immunogenic composition of
the invention is administered to a subject as a vaccine (e.g., to
prevent or attenuate a disease (e.g., allergic disease (e.g., by
providing to the subject total or partial immunity against the
disease or the total or partial attenuation (e.g., suppression) of
a sign, symptom or condition of the disease (e.g., sign, symptom or
condition of an allergic disease)))).
[0050] As used herein, the term "adjuvant" refers to any substance
that can stimulate an immune response. Some adjuvants can cause
activation of a cell of the immune system (e.g., an adjuvant can
cause an immune cell to produce and secrete a cytokine). Examples
of adjuvants that can cause activation of a cell of the immune
system include, but are not limited to, nanoemulsion formulations
described herein, saponins purified from the bark of the Q.
saponaria tree, such as QS21 (a glycolipid that elutes in the 21st
peak with HPLC fractionation; Aquila Biopharmaceuticals, Inc.,
Worcester, Mass.); poly(di(carboxylatophenoxy)phosphazene (PCPP
polymer; Virus Research Institute, USA); derivatives of
lipopolysacchandes such as monophosphoryl lipid A (MPL; Ribi
ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide
(MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a
glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin,
Switzerland); and Leishmania elongation factor (a purified
Leishmania protein: Corixa Corporation, Seattle, Wash.).
Traditional adjuvants are well known in the art and include, for
example, aluminum phosphate or hydroxide salts ("alum").
[0051] As used herein, the term "an amount effective to induce an
immune response" (e.g., of a composition for inducing an immune
response comprising a nanoemulsion formulated for injectable
administration), refers to the dosage level required (e.g., when
administered to a subject) to stimulate, generate and/or elicit an
immune response in the subject. An effective amount can be
administered in one or more administrations (e.g., via the same or
different route), applications or dosages and is not intended to be
limited to a particular formulation or administration route.
Accordingly, a "therapeutically effective amount" (e.g., of a
composition for inducing an immune response) refers to the dosage
level or amount of a composition required (e.g., when administered
to a subject (e.g., administered via injection)) to stimulate,
generate and/or elicit a therapeutic benefit in a subject. A
therapeutically effective amount can be administered in one or more
administrations (e.g., via the same or different route),
applications or dosages and is not intended to be limited to a
particular formulation or administration route.
[0052] As used herein, the term "under conditions such that said
subject generates an immune response" and grammatical equivalents
refer to any qualitative or quantitative induction, generation,
and/or stimulation of an immune response (e.g., innate or
acquired).
[0053] A used herein, the term "immune response" and grammatical
equivalents refer to a response by the immune system of a subject.
For example, immune responses include, but are not limited to, a
detectable alteration (e.g., increase) in Toll-like receptor (TLR)
activation, lymphokine (e.g., cytokine (e.g., Th1 or Th2 type
cytokines) or chemokine) expression and/or secretion, macrophage
activation, dendritic cell activation, T cell activation (e.g.,
CD4+ or CD8+ T cells), NK cell activation, and/or B cell activation
(e.g., antibody generation and/or secretion). Additional examples
of immune responses include binding of an immunogen (e.g., antigen
(e.g., immunogenic polypeptide)) to an MHC molecule and inducing a
cytotoxic T lymphocyte ("CTL") response, inducing a B cell response
(e.g., antibody production), and/or T-helper lymphocyte response,
and/or a delayed type hypersensitivity (DTH) response against the
antigen from which the immunogenic polypeptide is derived,
expansion (e.g., growth of a population of cells) of cells of the
immune system (e.g., T cells, B cells (e.g., of any stage of
development (e.g., plasma cells), and increased processing and
presentation of antigen by antigen presenting cells. An immune
response may be to immunogens that the subject's immune system
recognizes as foreign (e.g., non-self antigens from microorganisms
(e.g., pathogens), or self-antigens recognized as foreign). Thus,
it is to be understood that, as used herein, "immune response"
refers to any type of immune response, including, but not limited
to, innate immune responses (e.g., activation of Toll receptor
signaling cascade), cell-mediated immune responses (e.g., responses
mediated by T cells (e.g., antigen-specific T cells) and
non-specific cells of the immune system), and humoral immune
responses (e.g., responses mediated by B cells (e.g., via
generation and secretion of antibodies into the plasma, lymph,
and/or tissue fluids). The term "immune response" is meant to
encompass all aspects of the capability of a subject's immune
system to respond to antigens and/or immunogens (e.g., both the
initial response to an immunogen (e.g., a pathogen) as well as
acquired (e.g., memory) responses that are a result of an adaptive
immune response).
[0054] As used herein, the terms "toll receptors" and "TLRs" refer
to a class of receptors (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6,
TLR7, TLR8, TLR9, TLR10, TLR 11) that recognize special patterns of
pathogens, termed pathogen-associated molecular patterns (See,
e.g., Janeway and Medzhitov, (2002) Annu. Rev. Immunol. 20,
197-216). These receptors are expressed in innate immune cells
(e.g., neutrophils, monocytes, macrophages, dendritic cells) and in
other types of cells such as endothelial cells. Their ligands
include bacterial products such as LPS, peptidoglycans, and
lipopeptides. TLRs are receptors that bind to exogenous ligands and
mediate innate immune responses leading to the elimination of
invading microbes. The TLR-triggered signaling pathway leads to
activation of transcription factors including NFkB, which is
important for the induced expression of proinflammatory cytokines
and chemokines. TLRs also interact with each other. For example,
TLR2 can form functional heterodimers with TLR1 or TLR6. The TLR2/l
dimer has different ligand binding profile than the TLR2/6 dimer
(Ozinsky et al., 2000). In some embodiments, a nanoemulsion
adjuvant activates cell signaling through a TLR (e.g., TLR2 and/or
TLR4). Thus, methods described herein include a nanoemulsion
adjuvant composition (e.g., composition comprising NE adjuvant
optionally combined with one or more immunogens (e.g., protein
antigens or other antigen described herein)) that when administered
to a subject, activates one or more TLRs and stimulates an immune
response (e.g., innate and/or adaptive/acquired immune response) in
a subject. Such an adjuvant can activate TLRs (e.g., TLR2 and/or
TLR4) by, for example, interacting with TLRs (e.g., NE adjuvant
binding to TLRs) or activating any downstream cellular pathway that
occurs upon binding of a ligand to a TLR. NE adjuvants described
herein that activate TLRs can also enhance the availability or
accessibility of any endogenous or naturally occurring ligand of
TLRs. A NE adjuvant that activates one or more TLRs can alter
transcription of genes, increase translation of mRNA or increase
the activity of proteins that are involved in mediating TLR
cellular processes. For example, NE adjuvants described herein that
activate one or more TLRs (e.g., TLR2 and/or TLR4) can induce
expression of one or more cytokines (e.g., IL-8, IL-12p40, and/or
IL-23).
[0055] As used herein, the term "immunity" refers to protection
from disease (e.g., preventing or attenuating (e.g., suppression)
of a sign, symptom or condition of the disease) upon exposure to a
microorganism (e.g., pathogen) capable of causing the disease.
Immunity can be innate (e.g., non-adaptive (e.g., non-acquired)
immune responses that exist in the absence of a previous exposure
to an antigen) and/or acquired/adaptive (e.g., immune responses
that are mediated by B and T cells following a previous exposure to
antigen (e.g., that exhibit increased specificity and reactivity to
the antigen)).
[0056] As used herein, the terms "immunogen" and "antigen" are used
interchangeably to refer to an agent (e.g., a microorganism (e.g.,
bacterium, virus or fungus) and/or portion or component thereof
(e.g., protein, glycoprotein, lipoprotein, peptide, glycopeptide,
lipopeptide, toxoid, carbohydrate, tumor-specific antigen, etc.))
that is capable of eliciting an immune response in a subject. In
preferred embodiments, immunogens elicit immunity against the
immunogen (e.g., microorganism (e.g., pathogen or a pathogen
product), cancer and/or tumor, etc.) when administered in
combination with a nanoemulsion adjuvant formulation of the
invention comprising one or more antigens/immunogens together with
an adjuvant formulation comprising an emulsion delivery system in
combination with one or more immunostimulatory compounds (e.g., a
compound that stimulates the innate immune system (e.g., a
toll-like receptor antagonist (e.g., synthetic
oligodeoxynucleotides (ODN) (e.g., ODN containing immunostimulatory
CpG motifs (CpG))) or a saponin (e.g., a triterpene glycoside
saponin (e.g., QS-21))) formulated for administration (e.g., via
injectable route (e.g., intradermal, intramuscular, subcutaneously,
etc.), mucosal route (e.g., nasally or vaginally), or other route)
to a subject.
[0057] As used herein, "CpG oligonucleotide" "CpG ODN" and "CpG"
refer to an immunostimulatory nucleic acid containing at least one
cytosine-guanine dinucleotide sequence (e.g., a 5' cytidine
followed by 3' guanosine and linked by a phosphate bond) and which
activates the immune system. An "unmethylated CpG oligonucleotide"
is a nucleic acid molecule which contains an unmethylated
cytosine-guanine dinucleotide sequence (e.g., an unmethylated 5'
cytidine followed by 3' guanosine and linked by a phosphate bond)
and which activates the immune system. A "methylated CpG
oligonucleotide" is a nucleic acid which contains a methylated
cytosine-guanine dinucleotide sequence (e.g., a methylated 5'
cytidine followed by a 3' guanosine and linked by a phosphate bond)
and which activates the immune system. CpG oligonucleotides are
well known in the art and are described in, e.g., U.S. Pat. Nos.
6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and
6,339,068; PCT Publication No. WO 01/22990; PCT Publication No. WO
03/015711; US Publication No. 20030139364, each of which are hereby
incorporated by reference in its entirety.
[0058] As used herein, the term "pathogen product" refers to any
component or product derived from a pathogen including, but not
limited to, polypeptides, peptides, proteins, nucleic acids,
membrane fractions, and polysaccharides.
[0059] As used herein, the term "enhanced immunity" refers to an
increase in the level of adaptive and/or acquired immunity in a
subject to a given immunogen (e.g., microorganism (e.g., pathogen))
following administration of a composition (e.g., composition for
inducing an immune response of the present invention) relative to
the level of adaptive and/or acquired immunity in a subject that
has not been administered the composition (e.g., composition for
inducing an immune response of the present invention).
[0060] As used herein, the terms "purified" or "to purify" refer to
the removal of contaminants or undesired compounds from a sample or
composition. As used herein, the term "substantially purified"
refers to the removal of from about 70 to 90%, up to 100%, of the
contaminants or undesired compounds from a sample or
composition.
[0061] As used herein, the terms "administration" and
"administering" refer to the act of giving a composition of the
present invention (e.g., a composition for inducing an immune
response) to a subject. Exemplary routes of administration to the
human body include, but are not limited to, through the eyes
(ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs
(inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g.,
intravenously, subcutaneously, intraperitoneally, etc.), topically,
and the like. A preferred route of administration, according to the
invention, is via injection (e.g., intradermal, subcutaneous,
intramuscular, intravenous, intraosseous, intraperitoneal,
intrathecal, epidural, intracardiac, intraarticular,
intracavernous, micropenetrators, microdialysis, and/or
intravitreal).
[0062] As used herein, the terms "co-administration" and
"co-administering" refer to the administration of at least two
agent(s) (e.g., an immunogenic composition of the invention and one
or more other agents or therapies to a subject). In some
embodiments, the co-administration of two or more agents or
therapies is concurrent. In other embodiments, a first
agent/therapy is administered prior to a second agent/therapy. In
some embodiments, co-administration can be via the same or
different route of administration. Those of skill in the art
understand that the formulations and/or routes of administration of
the various agents or therapies used may vary. The appropriate
dosage for co-administration can be readily determined by one
skilled in the art. In some embodiments, when agents or therapies
are co-administered, the respective agents or therapies are
administered at lower dosages than appropriate for their
administration alone. Thus, co-administration is especially
desirable in embodiments where the co-administration of the agents
or therapies lowers the requisite dosage of a potentially harmful
(e.g., toxic) agent(s) (e.g., lowers the level of toxic
immunostimulatory agent (e.g., saponin) needed to be administered
to generate a desired immune response), and/or when
co-administration of two or more agents results in sensitization of
a subject to beneficial effects of one of the agents via
co-administration of the other agent. In other embodiments,
co-administration is preferable to elicit an immune response in a
subject to two or more different immunogens (e.g., antigens) at or
near the same time (e.g., when a subject is unlikely to be
available for subsequent administration of a second, third, or more
composition for inducing an immune response).
[0063] As used herein, the term "topically" refers to application
to the surface of the skin and/or mucosal cells and tissues (e.g.,
alveolar, buccal, lingual, masticatory, vaginal or nasal mucosa,
and other tissues and cells which line hollow organs or body
cavities). Topical administration may utilize a spray (e.g., a
nasal spray), a cream, or other viscous solution.
[0064] The terms "pharmaceutically acceptable" or
"pharmacologically acceptable," as used herein, refer to
compositions that do not substantially produce adverse reactions
(e.g., toxic, allergic or immunological reactions) when
administered to a subject.
[0065] As used herein, the term "pharmaceutically acceptable
carrier" refers to any of the standard pharmaceutical carriers
including, but not limited to, phosphate buffered saline solution,
water, and various types of wetting agents (e.g., sodium lauryl
sulfate), any and all solvents, dispersion media, coatings, sodium
lauryl sulfate, isotonic and absorption delaying agents,
disintrigrants (e.g., potato starch or sodium starch glycolate),
polyethylethe glycol, and the like. The compositions also can
include stabilizers and preservatives. Examples of carriers,
stabilizers and adjuvants have been described and are known in the
art (See e.g., Martin, Remington's Pharmaceutical Sciences, 15th
Ed., Mack Publ. Co., Easton. Pa. (1975), incorporated herein by
reference).
[0066] As used herein, the term "pharmaceutically acceptable salt"
refers to any salt (e.g., obtained by reaction with an acid or a
base) of a composition of the present invention that is
physiologically tolerated in the target subject. "Salts" of the
compositions of the present invention may be derived from inorganic
or organic acids and bases. Examples of acids include, but are not
limited to, hydrochloric, hydrobromic, sulfuric, nitric,
perchloric, fumaric, maleic, phosphoric, glycolic, lactic,
salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric,
methanesulfonic, ethanesulfonic, formic, benzoic, malonic,
sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the
like. Other acids, such as oxalic, while not in themselves
pharmaceutically acceptable, may be employed in the preparation of
salts useful as intermediates in obtaining the compositions of the
invention and their pharmaceutically acceptable acid addition
salts.
[0067] Examples of bases include, but are not limited to, alkali
metal (e.g., sodium) hydroxides, alkaline earth metal (e.g.,
magnesium) hydroxides, ammonia, and compounds of formula
NW.sub.4.sup.+, wherein W is C.sub.1-4 alkyl, and the like.
[0068] Examples of salts include, but are not limited to: acetate,
adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, chloride, bromide, iodide,
2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate,
persulfate, phenylpropionate, picrate, pivalate, propionate,
succinate, tartrate, thiocyanate, tosylate, undecanoate, and the
like. Other examples of salts include anions of the compounds of
the present invention compounded with a suitable cation such as
Na.sub.+, NH.sub.4.sup.+, and NW.sub.4.sup.+ (wherein W is a
C.sub.1-4 alkyl group), and the like. For therapeutic use, salts of
the compounds of the present invention are contemplated as being
pharmaceutically acceptable. However, salts of acids and bases that
are non-pharmaceutically acceptable may also find use, for example,
in the preparation or purification of a pharmaceutically acceptable
compound.
[0069] For therapeutic use, salts of the compositions of the
present invention are contemplated as being pharmaceutically
acceptable. However, salts of acids and bases that are
non-pharmaceutically acceptable may also find use, for example, in
the preparation or purification of a pharmaceutically acceptable
composition.
[0070] As used herein, the term "at risk for disease" refers to a
subject that is predisposed to experiencing a particular disease.
This predisposition may be genetic (e.g., a particular genetic
tendency to experience the disease, such as heritable disorders),
or due to other factors (e.g., environmental conditions, exposures
to detrimental compounds present in the environment, etc.). Thus,
it is not intended that the present invention be limited to any
particular risk (e.g., a subject may be "at risk for disease"
simply by being exposed to and interacting with other people), nor
is it intended that the present invention be limited to any
particular disease.
[0071] "Nasal application", as used herein, means applied through
the nose into the nasal or sinus passages or both. The application
may, for example, be done by drops, sprays, mists, coatings or
mixtures thereof applied to the nasal and sinus passages.
[0072] "Vaginal application", as used herein, means applied into or
through the vagina so as to contact vaginal mucosa. The application
may contact the urethra, cervix, fornix, uterus or other area
surrounding the vagina. The application may, for example, be done
by drops, sprays, mists, coatings, lubricants or mixtures thereof
applied to the vagina or surrounding tissue.
[0073] As used herein, the term "kit" refers to any delivery system
for delivering materials. In the context of immunogenic agents
(e.g., a composition comprising one or more antigens/immunogens
together with an adjuvant formulation comprising an emulsion
delivery system in combination with one or more immunostimulatory
compounds), such delivery systems include systems that allow for
the storage, transport, or delivery of immunogenic agents and/or
supporting materials (e.g., written instructions for using the
materials, a syringe and/or needle, etc.). For example, kits
include one or more enclosures (e.g., boxes) containing the
relevant immunogenic agents (e.g., nanoemulsions) and/or supporting
materials. As used herein, the term "fragmented kit" refers to
delivery systems comprising two or more separate containers that
each contain a subportion of the total kit components. The
containers may be delivered to the intended recipient together or
separately. For example, a first container may contain a
composition comprising one or more antigens/immunogens together
with an adjuvant formulation comprising an emulsion delivery system
in combination with one or more immunostimulatory compounds for a
particular use, while a second container contains a second agent
(e.g., a syringe and/or needle). Indeed, any delivery system
comprising two or more separate containers that each contains a
subportion of the total kit components are included in the term
"fragmented kit." In contrast, a "combined kit" refers to a
delivery system containing all of the components of an immunogenic
agent needed for a particular use in a single container (e.g., in a
single box housing each of the desired components). The term "kit"
includes both fragmented and combined kits.
DETAILED DESCRIPTION OF THE INVENTION
[0074] The present invention provides methods and compositions for
the stimulation of immune responses. In particular, the invention
provides methods and compositions for enhancing an immune response
to one or more antigens. Compositions and methods of the invention
are useful for the treatment and/or prevention of microbial
infections, such as infections caused by bacteria, viruses, fungi
and parasites. Compositions and methods of the invention include
one or more antigens/immunogens together with an adjuvant
formulation comprising an emulsion in combination with one or more
immunostimulatory compounds (e.g., a compound that stimulates the
innate immune system (e.g., a toll-like receptor antagonist (e.g.,
synthetic oligodeoxynucleotides (ODN)))) that enhance immune
responses to the one or more antigens/immunogens when administered
to a subject. Compositions and methods of the invention find use
in, among other things, clinical (e.g. therapeutic and preventative
medicine (e.g., vaccination)) and research applications.
[0075] In general, an immune response is generated to an antigen
through the interaction of the antigen with the cells of the immune
system. Immune responses may be broadly categorized into two
categories: humoral and cell mediated immune responses (e.g.,
traditionally characterized by antibody and cellular effector
mechanisms of protection, respectively). These categories of
response have been termed Th1-type responses (cell-mediated
response), and Th2-type immune responses (humoral response).
[0076] Stimulation of an immune response can result from a direct
or indirect response of a cell or component of the immune system to
an intervention (e.g., exposure to an immunogen). Immune responses
can be measured in many ways including activation, proliferation or
differentiation of cells of the immune system (e.g., B cells, T
cells, dendritic cells, APCs, macrophages. NK cells, NKT cells
etc.); up-regulated or down-regulated expression of markers and
cytokines; stimulation of IgA, IgM, or IgG titer; splenomegaly
(including increased spleen cellularity); hyperplasia and mixed
cellular infiltrates in various organs. Other responses, cells, and
components of the immune system that can be assessed with respect
to immune stimulation are known in the art.
[0077] An immune response refers to any qualitative or quantitative
induction, generation, and/or stimulation of an immune response in
a subject that is measurable. An immune response may be an innate
immune response (e.g., non-adaptive (e.g., non-acquired) immune
response that exists in the absence of a previous exposure to an
antigen) or an acquired/adaptive immune response (e.g., immune
response that is mediated by B and T cells following an exposure to
antigen (e.g., that exhibit increased specificity and reactivity to
the antigen)). In a preferred embodiment, an immune response
generated by an immunogenic composition of the invention provides
protection from disease (e.g., preventing or attenuating (e.g.,
suppression) of a sign, symptom or condition of the disease) upon
exposure to a microorganism (e.g., pathogen) capable of causing the
disease (e.g., from which an antigen/immunogen used in the
immunogenic composition of the invention is derived). Such
protection and/or prevention may be referred to as immunity.
[0078] The invention is not limited to any particular immune
response. Immune responses include, but are not limited to, a
detectable alteration (e.g., increase) in Toll-like receptor (TLR)
activation, lymphokine (e.g., cytokine (e.g., Th1 or Th2 type
cytokines) or chemokine) expression and/or secretion, macrophage
activation, dendritic cell activation, T cell activation (e.g.,
CD4+ or CD8+ T cells), NK cell activation, and/or B cell activation
(e.g., antibody generation and/or secretion). Additional examples
of immune responses include binding of an immunogen (e.g., antigen
(e.g., immunogenic polypeptide)) to an MHC molecule and inducing a
cytotoxic T lymphocyte ("CTL") response, inducing a B cell response
(e.g., antibody production), and/or T-helper lymphocyte response,
and/or a delayed type hypersensitivity (DTH) response against the
antigen from which an antigen/immunogen is derived, expansion
(e.g., growth of a population of cells) of cells of the immune
system (e.g., T cells, B cells (e.g., of any stage of development
(e.g., plasma cells), and increased processing and presentation of
antigen by antigen presenting cells. An immune response may be to
immunogens that the subject's immune system recognizes as foreign
(e.g., non-self antigens from microorganisms (e.g., pathogens), or
self-antigens recognized as foreign. Immune responses include, but
are not limited to, innate immune responses (e.g., activation of
Toll receptor signaling cascade), cell-mediated immune responses
(e.g., responses mediated by T cells (e.g., antigen-specific T
cells) and non-specific cells of the immune system), and humoral
immune responses (e.g., responses mediated by B cells (e.g., via
generation and secretion of antibodies into the plasma, lymph,
and/or tissue fluids). Immune responses encompass all aspects of
the capability of a subject's immune system to respond to antigens
and/or immunogens (e.g., both the initial response to an immunogen
(e.g., a pathogen) as well as acquired (e.g., memory) responses
that are a result of an adaptive immune response).
[0079] In a preferred embodiment, an immunogenic compositions of
the invention comprising one or more antigens/immunogens together
with an adjuvant formulation comprising an emulsion in combination
with one or more immunostimulatory compounds induces an immune
response to the one or more antigen/immunogens in a subject that is
greater than the immune response induced in a subject to the one or
more antigens/immunogens when the one or more antigens/immunogens
are administered with only a single component of the immunogenic
composition (e.g., when the one or more antigens/immunogens are
administered with only the emulsion (e.g., a nanoemulsion described
herein): or when the one or more antigens/immunogens are
administered with only the immunostimulatory compound (e.g., a
compound that stimulates the innate immune system (e.g., a
toll-like receptor antagonist (e.g., synthetic
oligodeoxynucleotides (ODN))))) (See, e.g., Example 1). In a
further preferred embodiment, the immune response induced in a
subject when an immunogenic compositions of the invention
comprising one or more antigens/immunogens together with an
adjuvant formulation comprising an emulsion in combination with one
or more immunostimulatory compounds is administered to the subject
is a synergistic immune response (e.g., synergistic compared to
when the one or more antigens/immunogens are administered with only
the emulsion (e.g., a nanoemulsion described herein), or when the
one or more antigens/immunogens are administered with only the
immunostimulatory compound (e.g., a compound that stimulates the
innate immune system (e.g., a toll-like receptor antagonist (e.g.,
synthetic oligodeoxynucleotides (ODN))))) (See, e.g., Example
1).
[0080] As described herein, an immune response induced may be an
innate immune response and/or an adaptive/acquired immune
response.
[0081] Host innate immune responses enable a host to differentiate
self from pathogen and provide a rapid inflammatory response,
including production of cytokines and chemokines, elaboration of
effector molecules, such as NO, and interactions with the adaptive
immune response (See, e.g., Janeway and Medzhitov, (2002) Annu.
Rev. Immunol. 20, 197-216). Molecular understanding of innate
immunity in humans evolved the mid-1990s when the Drosophila
protein Toll was shown to be critical for defending flies against
fungal infections (See, e.g., Lemaitre et al., (1996). Cell 86,
973-983). The human Toll-like receptor (TLR) family includes at
least ten receptors that play important roles in innate immunity
(See, e.g., Akira et al., (2006) Cell 124, 783-801; Beutler et al.,
(2006) Annu. Rev. Immunol. 24, 353-380; and Takeda et al., (2003).
Annu. Rev. Immunol. 21, 335-376).
[0082] In general, TLRs recognize and respond to diverse microbial
molecules and enable the innate immune system to discriminate among
groups of pathogens and to induce an appropriate cascade of
effector responses. Individual TLRs recognize a distinct repertoire
of conserved molecules (e.g., microbial products). For example,
well-characterized receptor-ligand pairs include TLR4 and LPS
(lipopolysaccharide), TLR5 and flagellin, TLR1/TLR2/TLR6 and
lipoproteins, and TLR3/TLR7/TLR8/TLR9 and different nucleic acid
motifs. Collectively, the family of TLRs allows a host's innate
immune system to detect the presence of foreign molecules (e.g.,
microbial products of most microbial pathogens or other
substances).
[0083] TLRs are classified as members of the IL-1R (IL-1 receptor)
superfamily on the basis of a shared cytoplasmic region known as
the TIR (Toll/IL-IR) domain. The extracellular portions of TLRs are
rather diverse, comprising varying numbers of leucine-rich repeats.
Following encounter with a microbe, TLRs trigger a complex cascade
of events that lead to the induction of a range of proinflammatory
genes (See, e.g., Yamamoto et al.. (2002) Nature 420, 324-329 (See,
e.g., Misch and Hawn, Clin Sci 2008, 114, 347-360)). Ligand binding
results in the recruitment of several molecules to the receptor
complex. These include TIR-domain-containing adaptor molecules such
as MyD88 (myeloid differentiation primary response gene 88),
TIRAP/Mal (TIR-domain-containing adapter/MyD88 adaptor-like),
TICAM1/TRIF (TIR-domain-containing adaptor molecule
1/TIR-domain-containing adaptor-inducing interferon b) and TRAM
(TRIF-related adaptor molecule). Further recruitment of molecules
includes IRAKs (IL-1R-associated kinases (IRAK1, 2, 3 (M) and 4))
as well as TRAF6 (TNF receptor-associated factor 6). IRAK1 and
TRAF6 then dissociate and bind another complex that comprises TAK1
(TGF (transforming growth factor)-b-activated kinase 1) and TAB1, 2
and 3 (TAK-1-binding proteins 1, 2 and 3). TAK1 then activates IKK
(IkB (inhibitor of NF-kB (nuclear factor kB)) kinase). The activity
of this complex is regulated by IKKg (also known as NEMO (NF-kB
essential modulator)). IKK-mediated phosphorylation of IkB leads to
its degradation, allowing NF-kB to translocate to the nucleus and
promote the transcription of multiple proinflammatory genes,
including TNF, IL-1b and IL-6.
[0084] TLR activation by pathogens, or by molecules derived
therefrom, induces intracellular signaling that primarily results
in activation of the transcription factor NF-kB (See, e.g., Beg.
2002, Trends Immunol. 2002 23 509-12.) and modulation of cytokine
production. However, a series of other pathways can also be
triggered, including p38 mitogen activated kinase, c-Jun-N-terminal
kinase and extracellular signal related kinase pathways (See, e.g.,
Flohe, et al., 2003, J Immunol, 170 2340-2348; Triantafilou &
Triantafilou, 2002, Trends Immunol, 23 301-304). The patterns of
gene expression induced by ligation of the different TLRs are
distinct but often overlap. For instance a large proportion of the
genes upregulated by TLR3 agonists and double stranded RNA are also
upregulated by TLR4 agonists and LPS (See, e.g., Doyle et al.,
2002, Immunity, 17 251-263). TLR4 activation by LPS in macrophages
results in TNF-.alpha., IL-12 IL-1P, RANTES and MIP1.beta.
secretion (See, e.g., Flohe et al., supra; Jones et al., 2002, J
Leukoc Biol, 69 1036-1044).
[0085] Adaptive/acquired immune responses are broadly categorized
into two categories: humoral and cell mediated immune responses
(e.g., traditionally characterized by antibody and cellular
effector mechanisms of protection, respectively). These categories
of response have been termed Th1-type responses (cell-mediated
response), and Th2-type immune responses (humoral response).
[0086] Stimulation of an immune response can result from a direct
or indirect response of a cell or component of the immune system to
an intervention (e.g., exposure to an antigen/immunogen (e.g., an
antigen combined with an adjuvant formulation of the invention
comprising an emulsion in combination with one or more
immunostimulatory compounds (e.g., a compound that stimulates an
innate immune response (e.g., a toll-like receptor antagonist
(e.g., synthetic oligodeoxynucleotides (ODN)))). Immune responses
can be measured in many ways including activation, proliferation or
differentiation of cells of the immune system (e.g., B cells, T
cells, dendritic cells, APCs, macrophages. NK cells, NKT cells
etc.); up-regulated or down-regulated expression of markers and
cytokines; stimulation of IgA. IgM, or IgG titer; splenomegaly
(including increased spleen cellularity); hyperplasia and mixed
cellular infiltrates in various organs.
[0087] Although an understanding of the mechanism is not necessary
to practice the present invention and the present invention is not
limited to any particular mechanism of action, in some embodiments,
immunogenic compositions and methods of the present invention
(e.g., comprising one or more antigens and an adjuvant formulation
comprising an emulsion in combination with one or more
immunostimulatory compounds) induce an additive or a more than
additive expression and/or secretion of cytokines (e.g., by
macrophages, dendritic cells and/or CD4+ T cells) when administered
to a subject when compared to when the one or more
antigens/immunogens are administered with only the emulsion (e.g.,
a nanoemulsion described herein); or when the one or more
antigens/immunogens are administered with only the
immunostimulatory compound (e.g., a compound that stimulates the
innate immune system (e.g., a toll-like receptor antagonist (e.g.,
synthetic oligodeoxynucleotides (ODN)))). Modulation of expression
of a particular cytokine can occur locally or systemically. It is
known that cytokine profiles can determine T cell regulatory and
effector functions in immune responses. In some embodiments,
Th1-type cytokines are induced, and thus, the immunostimulatory
compositions of the present invention promote (and can be used to
promote) a Th1 type antigen-specific immune response including
cytotoxic T-cells.
[0088] Cytokines play a role in directing the T cell response.
Helper (CD4+) T cells orchestrate the immune response of mammals
through production of soluble factors that act on other immune
system cells, including B and other T cells. Most mature CD4+ T
helper cells express one of two cytokine profiles: Th1 or Th2.
Th1-type CD4+ T cells secrete IL-2, IL-3, IFN-.gamma., GM-CSF and
high levels of TNF-.alpha.. Th2 cells express IL-3, IL-4, IL-5,
IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF-.alpha.. Th1
type cytokines promote both cell-mediated immunity, and humoral
immunity that is characterized by immunoglobulin class switching to
IgG2a in mice and IgG1 in humans. Th1 responses may also be
associated with delayed-type hypersensitivity and autoimmune
disease. Th2 type cytokines induce primarily humoral immunity and
induce class switching to IgG1 and IgE. The antibody isotypes
associated with Th1 responses generally have neutralizing and
opsonizing capabilities whereas those associated with Th2 responses
are associated more with allergic responses.
[0089] Several factors have been shown to influence skewing of an
immune response towards either a Th1 or Th2 type response, the best
characterized regulators are cytokines. IL-12 and IFN-.gamma.f are
positive Th1 and negative Th2 regulators. IL-12 promotes
IFN-.gamma. production, and IFN-.gamma. provides positive feedback
for IL-12. IL-4 and IL-10 appear important for the establishment of
the Th2 cytokine profile and to down-regulate Th cytokine
production.
[0090] In some embodiments, immunogenic compositions and methods of
the invention stimulate a Th-17 immune response (e.g., comprising
expression and/or secretion of IL-17).
[0091] In some preferred embodiments, the present invention
provides immunogenic compositions and methods of using the same to
stimulate a Th1-type immune response in a subject comprising
administering to a subject an immunogenic composition described
herein.
[0092] The invention is not limited by the type of
immunostimulatory molecule/compound used in an immunogenic
composition of the invention (e.g., in an adjuvant formulation
comprising an emulsion in combination with one or more
immunostimulatory agents/compounds). Indeed, a variety of a
immunostimulatory molecules/compounds may be used including, but
not limited to, the following. In some embodiments, monophosphoryl
lipid A (e.g., in particular 3-de-O-acylated monophosphoryl lipid A
(3D-MPL)), is used. 3D-MPL is a well-known adjuvant manufactured by
Ribi Immunochem, Montana. Chemically it is often supplied as a
mixture of 3-de-O-acylated monophosphoryl lipid A with either 4, 5,
or 6 acylated chains. In some embodiments, diphosphoryl lipid A,
and 3-O-deacylated variants thereof are used. Each of these
immunogens can be purified and prepared by methods described in GB
2122204B, hereby incorporated by reference in its entirety. Other
purified and synthetic lipopolysaccharides have been described
(See, e.g., U.S. Pat. No. 6,005,099 and EP 0 729 473; Hilgers et
al., 1986, Int. Arch. Allergy. Immunol., 79(4):392-6; Hilgers et
al., 1987, Immunology, 60(1):141-6; and EP 0 549 074, each of which
is hereby incorporated by reference in its entirety). In some
embodiments. 3D-MPL is used in the form of a particulate
formulation (e.g., having a small particle size less than 0.2 .mu.m
in diameter, described in EP 0 689 454, hereby incorporated by
reference in its entirety). In some embodiments, saponins are used
in an adjuvant formulation of an immunogenic composition of the
present invention. Saponins are well known adjuvants (See, e.g.,
Lacaille-Dubois and Wagner (1996) Phytomedicine vol 2 pp 363-386).
Examples of saponins include Quil A (derived from the bark of the
South American tree Quillaja Saponaria Molina), and fractions
thereof (See, e.g., U.S. Pat. No. 5,057,540; Kensil, Crit Rev Ther
Drug Carrier Syst, 1996, 12 (1-2):1-55; and EP 0 362 279, each of
which is hereby incorporated by reference in its entirety). Also
useful in the present invention are the haemolytic saponins QS7,
QS17, and QS21 (e.g., HPLC purified fractions of Quil A; See, e.g.,
Kensil et al. (1991). J. Immunology 146,431-437, U.S. Pat. No.
5,057,540; WO 96/33739; WO 96/11711 and EP 0 362 279, each of which
is hereby incorporated by reference in its entirety). Also useful
are combinations of QS21 and polysorbate or cyclodextrin (See,
e.g., WO 99/10008, hereby incorporated by reference in its
entirety). In some embodiments, an immunogenic oligonucleotide
containing unmethylated CpG dinucleotides ("CpG") is used as an
adjuvant in the present invention. CpG is an abbreviation for
cytosine-guanosine dinucleotide motifs present in DNA. CpG is known
in the art as being an adjuvant when administered by both systemic
and mucosal routes (See, e.g., WO 96/02555, EP 468520, Davis et
al., J. Immunol, 1998, 160(2):870-876; McCluskie and Davis, J.
Immunol., 1998, 161(9):4463-6; and U.S. Pat. App. No. 20050238660,
each of which is hereby incorporated by reference in its entirety).
For example, in some embodiments, the immunostimulatory sequence is
Purine-Purine-C-G-pyrimidine-pyrimidine: wherein the CG motif is
not methylated.
[0093] Although an understanding of the mechanism is not necessary
to practice the present invention and the present invention is not
limited to any particular mechanism of action, in some embodiments,
the presence of one or more CpG oligonucleotides activate various
immune subsets including natural killer cells (which produce
IFN-.gamma.) and macrophages. In some embodiments, CpG
oligonucleotides are formulated into an immunogenic composition of
the present invention for inducing an immune response. In some
embodiments, a free solution of CpG is co-administered together
with an antigen (e.g., present within a NE solution). In some
embodiments, a NE plus CpG is co-administered together with an
antigen. In some embodiments, a CpG oligonucleotide is covalently
conjugated to an antigen (See, e.g., WO 98/16247, hereby
incorporated by reference), or formulated with a carrier such as
aluminium hydroxide (See, e.g., Brazolot-Millan et al., Proc. Natl.
Acad Sci., USA, 1998, 95(26). 15553-8).
[0094] In some embodiments, adjuvants such as Complete Freunds
Adjuvant and Incomplete Freunds Adjuvant, cytokines (e.g.,
interleukins (e.g., IL-2, IFN-.gamma., IL-4, etc.), macrophage
colony stimulating factor, tumor necrosis factor, etc.), detoxified
mutants of a bacterial ADP-ribosylating toxin such as a cholera
toxin (CT), a pertussis toxin (PT), or an E. Coli heat-labile toxin
(LT), particularly LT-K63 (where lysine is substituted for the
wild-type amino acid at position 63) LT-R72 (where arginine is
substituted for the wild-type amino acid at position 72), CT-S109
(where serine is substituted for the wild-type amino acid at
position 109), and PT-K9/G129 (where lysine is substituted for the
wild-type amino acid at position 9 and glycine substituted at
position 129) (See, e.g., WO93/13202 and WO92/19265, each of which
is hereby incorporated by reference), and/or other immunogenic
compounds/substances (e.g., that stimulate an immune response) are
used in or in combination with an immunogenic composition
comprising an adjuvant formulation comprising an emulsion in
combination with one or more immunostimulatory compounds of the
present invention.
[0095] Additional examples of adjuvants that may be used in or in
combination with an immunogenic composition of the invention (e.g.,
an immunogenic composition comprising an adjuvant formulation
comprising an emulsion in combination with one or more
immunostimulatory compounds) include
poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus
Research Institute, USA); derivatives of lipopolysaccharides such
as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc.,
Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and
threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine
disaccharide related to lipid A; OM Pharma SA, Meyrin,
Switzerland); and Leishmania elongation factor (a purified
Leishmania protein; Corixa Corporation, Seattle, Wash.).
[0096] Adjuvants may be added to a composition comprising a
nanoemulsion adjuvant and an immunogen, or, the adjuvant may be
formulated with carriers, for example liposomes, or metallic salts
(e.g., aluminium salts (e.g., aluminium hydroxide)) prior to
combining with or co-administration with a composition comprising a
nanoemulsion adjuvant and an immunogen.
[0097] In some embodiments, an immunogenic composition comprising a
nanoemulsion adjuvant and an immunogen comprises a single
additional immunostimulatory compound/molecule and/or adjuvant. In
other embodiments, an immunogenic composition comprising a
nanoemulsion adjuvant and an immunogen comprises two or more
additional immunostimulatory compounds/molecules and/or adjuvants
(See, e.g., WO 94/00153; WO 95/17210; WO 96/33739: WO 98/56414; WO
99/12565: WO 99/11241; and WO 94/00153, each of which is hereby
incorporated by reference in its entirety).
[0098] In some embodiments, an immunogenic composition described
herein (e.g., comprising one or more antigens/immunogens together
with an adjuvant formulation comprising an emulsion in combination
with one or more immunostimulatory compounds) of the present
invention comprises one or more mucoadhesives (See, e.g., U.S. Pat.
App. No. 20050281843, hereby incorporated by reference in its
entirety). The present invention is not limited by the type of
mucoadhesive utilized. Indeed, a variety of mucoadhesives are
contemplated to be useful in the present invention including, but
not limited to, cross-linked derivatives of poly(acrylic acid)
(e.g., carbopol and polycarbophil), polyvinyl alcohol, polyvinyl
pyrollidone, polysaccharides (e.g., alginate and chitosan),
hydroxypropyl methylcellulose, lectins, fimbrial proteins, and
carboxymethylcellulose. In some embodiments, one or more components
of a NE adjuvant of the immunogenic composition function as a
mucoadhesive (e.g., individually, or in combination with other
components of the NE adjuvant). Although an understanding of the
mechanism is not necessary to practice the present invention and
the present invention is not limited to any particular mechanism of
action, in some embodiments, use of a mucoadhesive (e.g., in an
immunogenic composition of the invention) enhances induction of an
immune response (e.g., an innate and/or adaptive immune response)
in a subject (e.g., a subject administered an immunogenic
composition of the present invention) due to an increase in
duration and/or amount of exposure to the immunogenic composition
that a subject experiences when a mucoadhesive is used compared to
the duration and/or amount of exposure to the immunogenic
composition in the absence of using the mucoadhesive.
[0099] In some embodiments, one or more components of the
immunogenic composition function to delay the release of the
antigen/immunogen component of the composition. Although an
understanding of the mechanism is not necessary to practice the
present invention and the present invention is not limited to any
particular mechanism of action, in some embodiments, delayed or
time release (e.g., using particle technology and encapsulation
technology known in the art enhances induction of an immune
response (e.g., an innate and/or adaptive immune response) in a
subject (e.g., a subject administered an immunogenic composition of
the present invention) due to an increase in duration and/or amount
of exposure to the immunogenic composition that a subject
experiences compared to the duration and/or amount of exposure to
the immunogenic composition in the absence of the delayed and/or
timed release component.
[0100] In some embodiments, a composition of the present invention
may comprise sterile aqueous preparations. Acceptable vehicles and
solvents include, but are not limited to, water, Ringer's solution,
phosphate buffered saline and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed
mineral or non-mineral oil may be employed including synthetic
mono-ordi-glycerides. In addition, fatty acids such as oleic acid
find use in the preparation of injectables. Carrier formulations
suitable for mucosal, subcutaneous, intramuscular, intraperitoneal,
intravenous, or administration via other routes may be found in
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa.
[0101] An immunogenic composition of the present invention can be
used therapeutically (e.g., to enhance an immune response) or as a
prophylactic (e.g., for immunization (e.g., to prevent signs or
symptoms of disease)). An immunogenic composition of the present
invention can be administered to a subject via a number of
different delivery routes and methods.
[0102] For example, an immunogenic composition of the present
invention can be administered to a subject (e.g., mucosally (e.g.,
nasal mucosa, vaginal mucosa, etc.)) by multiple methods,
including, but not limited to: being suspended in a solution and
applied to a surface; being suspended in a solution and sprayed
onto a surface using a spray applicator; being mixed with a
mucoadhesive and applied (e.g., sprayed or wiped) onto a surface
(e.g., mucosal surface); being placed on or impregnated onto a
nasal and/or vaginal applicator and applied; being applied by a
controlled-release mechanism; being applied as a liposome; or being
applied on a polymer.
[0103] In some embodiments, compositions of the present invention
are administered mucosally (e.g., using standard techniques; See,
e.g., Remington: The Science and Practice of Pharmacy. Mack
Publishing Company, Easton, Pa., 19th edition, 1995 (e.g., for
mucosal delivery techniques, including intranasal, pulmonary,
vaginal and rectal techniques), as well as European Publication No.
517,565 and Illum et al., J. Controlled Rel., 1994, 29:133-141
(e.g., for techniques of intranasal administration), each of which
is hereby incorporated by reference in its entirety).
Alternatively, the compositions of the present invention may be
administered dermally or transdermally, using standard techniques
(See, e.g., Remington: The Science arid Practice of Pharmacy, Mack
Publishing Company. Easton, Pa., 19th edition, 1995). The present
invention is not limited by the route of administration.
[0104] In some embodiments, an immunogenic composition of the
present invention is used to protect and/or treat a subject
susceptible to, or suffering from, a disease by means of
administering the composition via injection (e.g., via intradermal,
subcutaneous, intramuscular, intravenous, intraosseous,
intraperitoneal, intrathecal, epidural, intracardiac,
intraarticular, intracavernous, and/or intravitreal route). Methods
of systemic administration include conventional syringes and
needles, or devices designed for ballistic delivery (See, e.g., WO
99/27961, hereby incorporated by reference), or needleless pressure
liquid jet device (See, e.g., U.S. Pat. Nos. 4,596,556; 5,993,412,
each of which are hereby incorporated by reference), or transdermal
patches (See, e.g., WO 97/48440: WO 98/28037, each of which are
hereby incorporated by reference). In some embodiments, the present
invention provides a delivery device for systemic administration,
pre-filled with an immunogenic composition of the present
invention.
[0105] In some embodiments, an immunogenic composition of the
present invention is used to protect or treat a subject susceptible
to, or suffering from, disease by means of administering a
composition of the present invention via a mucosal route (e.g., an
oral/alimentary or nasal route). Alternative mucosal routes include
intravaginal and intra-rectal routes. In preferred embodiments of
the present invention, a nasal route of administration is used,
termed "intranasal administration" or "intranasal vaccination"
herein. Methods of intranasal vaccination are well known in the
art, including the administration of a droplet or spray form of the
vaccine into the nasopharynx of a subject to be immunized. In some
embodiments, a nebulized or aerosolized immunogenic composition is
provided. Enteric formulations such as gastro resistant capsules
for oral administration, suppositories for rectal or vaginal
administration also form part of this invention. Compositions of
the present invention may also be administered via the oral route.
Under these circumstances, an immunogenic composition may comprise
a pharmaceutically acceptable excipient and/or include alkaline
buffers, or enteric capsules. Formulations for nasal delivery may
include those with dextran or cyclodextran and saponin as an
adjuvant.
[0106] An immunogenic composition may also be administered via a
vaginal route. In such cases, an immunogenic composition may
comprise pharmaceutically acceptable excipients and/or emulsifiers,
polymers (e.g., CARBOPOL), and other known stabilizers of vaginal
creams and suppositories. In some embodiments, compositions of the
present invention are administered via a rectal route. In such
cases, an immunogenic composition may comprise excipients and/or
waxes and polymers known in the art for forming rectal
suppositories.
[0107] In some embodiments, the same route of administration (e.g.,
mucosal administration) is chosen for both a priming and boosting
vaccination. In some embodiments, multiple routes of administration
are utilized (e.g., at the same time, or, alternatively,
sequentially) in order to stimulate an immune response (e.g., using
one or more immunogenic compositions of the present invention).
[0108] For example, in some embodiments, an immunogenic composition
is administered to a mucosal surface of a subject in either a
priming or boosting vaccination regime. Alternatively, in some
embodiments, an immunogenic composition is administered
systemically in either a priming or boosting vaccination regime. In
some embodiments, an immunogenic composition is administered to a
subject in a priming vaccination regimen via mucosal administration
and a boosting regimen via systemic administration. In some
embodiments, an immunogenic composition is administered to a
subject in a priming vaccination regimen via systemic
administration and a boosting regimen via mucosal administration.
Examples of systemic routes of administration include, but are not
limited to, a parenteral, intramuscular, intradermal, transdermal,
subcutaneous, intraperitoneal or intravenous administration. An
immunogenic composition may be used for both prophylactic and
therapeutic purposes.
[0109] NEs have not been shown to be inflammatory when placed on
the skin or mucous membranes in studies on animals and in humans.
Thus, although an understanding of the mechanism is not necessary
to practice the present invention and the present invention is not
limited to any particular mechanism of action, in some embodiments,
a composition comprising an immunogenic composition of the present
invention (e.g., comprising an adjuvant formulation comprising an
emulsion in combination with one or more immunostimulatory
compounds) acts to transport and/or present antigen/immunogen to
the immune system (e.g., to antigen presenting cells of the immune
system). In a preferred embodiment, a composition comprising an
immunogenic composition of the present invention (e.g., comprising
one or more antigens and an adjuvant formulation comprising an
emulsion in combination with one or more immunostimulatory
compounds) acts to transport and/or present antigen to the immune
system (e.g., to antigen presenting cells of the immune system) in
a greater way or in a synergistic way compared to when the one or
more antigens are administered with only the emulsion (e.g., a
nanoemulsion described herein); or when the one or more antigens
are administered with only the immunostimulatory compound (e.g., a
compound that stimulates the innate immune system (e.g., a
toll-like receptor antagonist (e.g., synthetic
oligodeoxynucleotides (ODN)))) (See, e.g., Example 1). In some
embodiments, mucosal administration of an immunogenic composition
of the present invention generates mucosal (e.g., signs of mucosal
immunity (e.g., generation of IgA antibody titers)) as well as
systemic immunity. In some embodiments, mucosal administration of
immunogenic composition of the invention generates an innate immune
response (e.g., activates Toll-like receptor signaling and/or
activation of NF-kB) in a subject. Both cellular and humoral
immunity play a role in protection against multiple pathogens and
both can be induced with immunogenic composition of the present
invention.
[0110] Accordingly, in a preferred embodiment, administration of an
immunogenic composition of the present invention primes, enables
and/or enhances induction of both humoral (e.g., development of
specific antibodies) and cellular (e.g., cytotoxic T lymphocyte)
immune responses (e.g., against a pathogen). In some embodiments,
an immunogenic composition of the present invention is used in a
vaccine (e.g., as an immunostimulatory adjuvant (e.g., that elicits
and/or enhances immune responses (e.g., innate and or adaptive
immune responses) in a host administered the immunogenic
composition).
[0111] In some embodiments, the present invention provides
immunogenic compositions comprising an adjuvant formulation
comprising an emulsion in combination with one or more
immunostimulatory compounds (e.g., a compound that stimulates the
innate immune system (e.g., a toll-like receptor antagonist (e.g.,
synthetic oligodeoxynucleotides (ODN)))) that possesses greater
efficacy at eliciting immune responses (e.g., innate immune
responses and/or adaptive/acquired immune responses) than either
emulsion or the one or more immunostimulatory compounds can
individually. Although an understanding of a mechanism is not
necessary to practice the present invention, and the present
invention is not limited to any particular mechanism of action, in
some embodiments, one or more antigens of an immunogenic
composition of the invention are more readily internalized by
phagocytic cells (e.g., macrophages, dendritic cells, B cells,
etc.) or other cells compared to when the one or more
antigens/immunogens are administered with only the emulsion (e.g.,
a nanoemulsion described herein); or when the one or more
antigens/immunogens are administered with only the
immunostimulatory compound (e.g., leading to greater
internalization of the one or more antigens (e.g., by antigen
presenting cells), processing of antigen, and/or presentation of
antigen to B and/or T cells). Thus, in some embodiments, greater
internalization and/or processing of antigen and/or presentation of
antigen to B and/or T cells leads to stronger, more robust immune
responses (e.g., to an antigen administered in an immunogenic
composition of the invention.
[0112] In some embodiments, the present invention provides an
immunogenic composition that generates a desired immune response in
a subject administered the same (e.g., an adaptive immune
response). For example, in some embodiments, the present invention
provides an immunogenic composition that skew a host's immune
response away from Th2 type immune response and toward a Th1 type
immune response (e.g., based upon selection of the constituents of
an the immunogenic composition). In particular, conventional alum
based vaccines for a variety of diseases such as respiratory
syncitial virus (RSV), anthrax, and hepatitis B virus each lead to
a predominant Th2 type immune response in a subject administered
the vaccine (e.g., characterized by enhanced expression of Th2 type
cytokines and the production of IgG1 antibodies). However, an
immunogenic composition of the invention is able to, in one
embodiment, redirect the conventionally observed Th2 type immune
response in host subjects administered conventional vaccines. Thus,
in some embodiments, the present invention provides immunogenic
compositions and methods for skewing and/or redirecting a host's
immune response (e.g., away from Th2 type immune responses and
toward Th1 type immune responses) to one or a plurality of
immunogens/antigens. In some embodiments, skewing and/or
redirecting a host's immune response (e.g., away from Th2 type
immune responses and toward Th1 type immune responses) to one or a
plurality of immunogens/antigens comprises providing one or more
antigens (e.g., recombinant antigens, isolated and/or purified
antigens, and/or killed whole pathogens) that are historically
associated with generation of a Th2 type immune response when
administered to a subject (e.g., RSV antigen, hepatitis B virus
antigen, etc.), combining the one or more antigens with an
immunogenic composition of the invention, and administering the
immunogenic composition to a subject under conditions sufficient to
induce the desired immune response.
[0113] In some embodiments, the present invention provides an
immunogenic composition that reduces the number of booster
injections (e.g., of an antigen containing composition) required to
achieve a desired immune response (e.g., a protective immune
response (e.g., a memory immune response)). In some embodiments,
the present invention provides an immunogenic composition that
results in a higher proportion of recipients achieving
seroconversion and/or more consistent immune responses within a
population of subjects administered the immunogenic composition. In
some embodiments, the present invention provides immunogenic
compositions that are useful for selectively skewing adaptive
immunity toward Th1, Th2, or cytotoxic T cell responses (e.g.,
allowing effective immunization by distinct routes (e.g., such as
via mucosa or via injection)). In some embodiments, the present
invention provides immunogenic compositions that elicit optimal
responses in subjects in which most contemporary vaccination
strategies are not optimally effective (e.g., in very young and/or
very old populations). In some embodiments, the present invention
provides immunogenic compositions that provide efficacy and safety
needed for vaccination regimens that involve different delivery
routes and elicitation of distinct types of immunity. In some
embodiments, the present invention provides immunogenic
compositions that stimulate antibody responses and have little
toxicity and that can be utilized with a range of antigens for
which they provide adjuvanticity and the types of immune responses
they elicit. In some embodiments, the present invention provides
immunogenic compositions that meet global supply requirements
(e.g., in response to a pathogenic (e.g., influenza) pandemic).
Generation of Antibodies
[0114] An immunogenic composition of the invention can be used to
immunize a mammal, such as a mouse, rat, rabbit, guinea pig,
monkey, or human, to produce polyclonal antibodies. If desired, an
antigen can be conjugated to a carrier protein, such as bovine
serum albumin, thyroglobulin, keyhole limpet hemocyanin or other
carrier described herein. Depending on the host species, various
additional adjuvants can be used to increase the immunological
response. Such adjuvants include, but are not limited to, Freund's
adjuvant, mineral gels (e.g., aluminum hydroxide), and surface
active substances (e.g. lysolecithin, pluronic polyols, polyanions,
peptides, nanoemulsions described herein, keyhole limpet
hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG
(bacilli Calmette-Guerin) and Corynebacterium parvum are especially
useful.
[0115] Monoclonal antibodies can be prepared using any technique
which provides for the production of antibody molecules by
continuous cell lines in culture. These techniques include, but are
not limited to, the hybridoma technique, the human B cell hybridoma
technique, and the EBV hybridoma technique (See, e.g., Kohler et
al., Nature 256, 495 497, 1985; Kozbor et al., J. Immunol. Methods
81, 3142, 1985: Cote et al., Proc. Natl. Acad. Sci. 80, 2026 2030,
1983; Cole et al., Mol. Cell. Biol. 62, 109 120, 1984).
[0116] In addition, techniques developed for the production of
"chimeric antibodies," the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity, can be used (See, e.g.,
Morrison et al.. Proc. Natl. Acad. Sci. 81, 68516855, 1984;
Neuberger et al, Nature 312, 604 608, 1984; Takeda et al., Nature
314, 452 454, 1985). Monoclonal and other antibodies also can be
"humanized" to prevent a patient from mounting an immune response
against the antibody when it is used therapeutically. Such
antibodies may be sufficiently similar in sequence to human
antibodies to be used directly in therapy or may require alteration
of a few key residues. Sequence differences between rodent
antibodies and human sequences can be minimized by replacing
residues which differ from those in the human sequences by site
directed mutagenesis of individual residues or by grating of entire
complementarity determining regions.
[0117] Alternatively, humanized antibodies can be produced using
recombinant methods, as described below. Antibodies which
specifically bind to a particular antigen can contain antigen
binding sites which are either partially or fully humanized, as
disclosed in U.S. Pat. No. 5,565,332.
[0118] Alternatively, techniques described for the production of
single chain antibodies can be adapted using methods known in the
art to produce single chain antibodies which specifically bind to a
particular antigen. Antibodies with related specificity, but of
distinct idiotypic composition, can be generated by chain shuffling
from random combinatorial immunoglobin libraries (See, e.g.,
Burton, Proc. Natl. Acad. Sci. 88, 11120 23, 1991).
[0119] Single-chain antibodies also can be constructed using a DNA
amplification method, such as PCR, using hybridoma cDNA as a
template (See, e.g., Thirion et al., 1996, Eur. J. Cancer Prev. 5,
507-11). Single-chain antibodies can be mono- or bispecific, and
can be bivalent or tetravalent. Construction of tetravalent,
bispecific single-chain antibodies is taught, for example, in
Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63.
Construction of bivalent, bispecific single-chain antibodies is
taught, for example, in Mallender & Voss, 1994, J. Biol. Chem.
269, 199-206.
[0120] A nucleotide sequence encoding a single-chain antibody can
be constructed using manual or automated nucleotide synthesis,
cloned into an expression construct using standard recombinant DNA
methods, and introduced into a cell to express the coding sequence,
as described below. Alternatively, single-chain antibodies can be
produced directly using, for example, filamentous phage technology
(See, e.g., Verhaar et al., 1995, Int. J. Cancer 61, 497-501;
Nicholls et al., 1993, J. Immunol. Meth. 165, 81-91).
[0121] Antibodies can be produced by inducing in vivo production in
the lymphocyte population or by screening immunoglobulin libraries
or panels of highly specific binding reagents as disclosed in the
literature (See, e.g., Orlandi et al., Proc. Natl. Acad. Sci. 86,
3833 3837, 1989; Winter et al., Nature 349, 293 299, 1991).
[0122] Chimenc antibodies can be constructed as disclosed in WO
93/03151. Binding proteins which are derived from immunoglobulins
and which are multivalent and multispecific, such as the
"diabodies" described in WO 94/13804, also can be prepared.
Antibodies can be purified by methods well known in the art. For
example, antibodies can be affinity purified by passage over a
column to which the relevant antigen is bound. The bound antibodies
can then be eluted from the column using a buffer with a high salt
concentration.
[0123] Immunogenic compositions of the invention are not limited by
the type of nanoemulsion utilized. Any number of suitable
nanoemulsion compositions may be utilized in the immunogenic
compositions (e.g., vaccines) compositions of the present
invention, including, but not limited to, those disclosed in
Hamouda el al., J. Infect Dis., 180:1939 (1999); Hamouda and Baker,
J. Appl. Microbiol., 89:397 (2000); and Donovan el al., Antivir.
Chem. Chemother., 11:41 (2000). Preferred nanoemulsions of the
present invention are those that are non-toxic to animals. In
preferred embodiments, nanoemulsions utilized in the compositions
and methods of the present invention are stable, and do not
decompose even after long storage periods (e.g., one or more
years). Additionally, preferred emulsions maintain stability even
after exposure to high temperature and freezing. This is especially
useful if they are to be applied in extreme conditions (e.g.,
extreme heat or cold).
[0124] Some embodiments of the present invention employ an oil
phase containing ethanol. For example, in some embodiments, the
emulsions of the present invention contain (i) an aqueous phase and
(ii) an oil phase containing ethanol as the organic solvent and
optionally a germination enhancer, and (iii) TYLOXAPOL as the
surfactant (preferably 2-5%, more preferably 3%). This formulation
is highly efficacious for inactivation of pathogens and is also
non-irritating and non-toxic to mammalian subjects (e.g., and thus
can be used for administration to a mucosal surface).
[0125] In some other embodiments, the emulsions of the present
invention comprise a first emulsion emulsified within a second
emulsion, wherein (a) the first emulsion comprises (i) an aqueous
phase; and (ii) an oil phase comprising an oil and an organic
solvent; and (iii) a surfactant; and (b) the second emulsion
comprises (i) an aqueous phase; and (ii) an oil phase comprising an
oil and a cationic containing compound; and (iii) a surfactant.
[0126] Nanoemulsions.
[0127] As described herein, experiments conducted during
development of embodiments of the invention identified certain,
specific nanoemulsoin formulations that, when combined with other
immunostimulatory compounds/molecules, displayed synergistic
immunostimulatory properties (e.g., when administered to a subject
(See, e.g., Example 1)).
[0128] Emulsion formulations described herein are simply examples
to illustrate the variety of nanoemulsion adjuvants that find use
in the present invention. The present invention contemplates that
many variations of these formulations, as well as additional
nanoemulsions, find use in the methods of the present invention.
Candidate emulsions can be easily tested to determine if they are
suitable for use in the immunogenic compositions of the
invention.
[0129] In preferred embodiments of the present invention, emulsion
formulations are non-toxic (e.g., to humans, plants, or animals),
non-irritant (e.g., to humans, plants, or animals), and
non-corrosive (e.g., to humans, plants, or animals or the
environment), while retaining stability when mixed with other
agents (e.g., a composition comprising an immunogen (e.g.,
bacteria, fungi, viruses, and spores).
[0130] The nanoemulsion can comprise an aqueous phase, at least one
oil, at least one surfactant, and at least one solvent.
Nanoemulsions of the present disclosure may comprise the following
properties and components.
[0131] The nanoemulsion vaccine of the present disclosure comprises
droplets having an average diameter size of less than about 1000
nm, less than about 950 nm, less than about 900 nm, less than about
850 nm, less than about 800 nm, less than about 750 nm, less than
about 700 nm, less than about 650 nm, less than about 600 nm, less
than about 550 nm, less than about 500 nm, less than about 450 nm,
less than about 400 nm, less than about 350 nm, less than about 300
nm, less than about 250 nm, less than about 200 nm, less than about
150 nm, or any combination thereof. In one embodiment, the droplets
have an average diameter size greater than about 125 nm and less
than or equal to about 600 nm. In a different embodiment, the
droplets have an average diameter size greater than about 50 nm or
greater than about 70 nm, and less than or equal to about 125
nm.
[0132] Aqueous Phase. The aqueous phase can comprise any type of
aqueous phase including, but not limited to, water (e.g., H.sub.2O,
distilled water, purified water, water for injection, de-ionized
water, tap water) and solutions (e.g., phosphate buffered saline
(PBS) solution). In certain embodiments, the aqueous phase
comprises water at a pH of about 4 to 10, preferably about 6 to 8.
The water can be deionized (hereinafter "DiH.sub.2O"). In some
embodiments the aqueous phase comprises phosphate buffered saline
(PBS). The aqueous phase may further be sterile and pyrogen
free.
[0133] Organic Solvents. Organic solvents in the nanoemulsion
include, but are not limited to, C.sub.1-C.sub.12 alcohol, diol,
triol, dialkyl phosphate, tri-alkyl phosphate, such as tri-n-butyl
phosphate, semi-synthetic derivatives thereof, and combinations
thereof. In one aspect of the disclosed, the organic solvent is an
alcohol chosen from a nonpolar solvent, a polar solvent, a protic
solvent, or an aprotic solvent.
[0134] Suitable organic solvents include, but are not limited to,
ethanol, methanol, isopropyl alcohol, glycerol, medium chain
triglycerides, diethyl ether, ethyl acetate, acetone, dimethyl
sulfoxide (DMSO), acetic acid, n-butanol, butylene glycol,
perfumers alcohols, isopropanol, n-propanol, formic acid, propylene
glycols, glycerol, sorbitol, industrial methylated spirit,
triacetin, hexane, benzene, toluene, diethyl ether, chloroform.
1,4-dixoane, tetrahydrofuran, dichloromethane, acetone,
acetonitrile, dimethylformamide, dimethyl sulfoxide, formic acid,
semi-synthetic derivatives thereof, and any combination
thereof.
[0135] Oil Phase. The oil in the nanoemulsion can be any
cosmetically or pharmaceutically acceptable oil. The oil can be
volatile or non-volatile, and may be chosen from animal oil,
vegetable oil, natural oil, synthetic oil, hydrocarbon oils,
silicone oils, semi-synthetic derivatives thereof, and combinations
thereof.
[0136] Suitable oils include, but are not limited to, mineral oil,
squalene oil, flavor oils, silicon oil, essential oils, water
insoluble vitamins, Isopropyl stearate, Butyl stearate, Octyl
palmitate, Cetyl palmitate, Tridecyl behenate, Diisopropyl adipate,
Dioctyl sebacate, Menthyl anthranhilate, Cetyl octanoate, Octyl
salicylate, Isopropyl myristate, neopentyl glycol dicarpate cetols,
Ceraphyls.RTM., Decyl oleate, diisopropyl adipate, C.sub.12-15
alkyl lactates, Cetyl lactate, Lauryl lactate, Isostearvl
neopentanoate, Myristyl lactate. Isocetyl stearoyl stearate.
Octyldodecyl stearoyl stearate. Hydrocarbon oils, Isoparaffin,
Fluid paraffins, Isododecane, Petrolatum, Argan oil, Canola oil,
Chile oil, Coconut oil, corn oil, Cottonseed oil, Flaxseed oil,
Grape seed oil, Mustard oil, Olive oil, Palm oil, Palm kernel oil,
Peanut oil, Pine seed oil, Poppy seed oil. Pumpkin seed oil, Rice
bran oil, Safflower oil, Tea oil, Truffle oil, Vegetable oil,
Apricot (kernel) oil, Jojoba oil (Simmondsia chinensis seed oil),
Grapeseed oil, Macadamia oil, Wheat germ oil, Almond oil, Rapeseed
oil, Gourd oil, Soybean oil, Sesame oil. Hazelnut oil, Maize oil,
Sunflower oil, Hemp oil, Bois oil, Kuki nut oil. Avocado oil,
Walnut oil, Fish oil, berry oil, allspice oil, juniper oil, seed
oil, almond seed oil, anise seed oil, celery seed oil, cumin seed
oil, nutmeg seed oil, leaf oil, basil leaf oil, bay leaf oil,
cinnamon leaf oil, common sage leaf oil, eucalyptus leaf oil, lemon
grass leaf oil, melaleuca leaf oil, oregano leaf oil, patchouli
leaf oil, peppermint leaf oil, pine needle oil, rosemary leaf oil,
spearmint leaf oil, tea tree leaf oil, thyme leaf oil, wintergreen
leaf oil, flower oil, chamomile oil, clary sage oil, clove oil,
geranium flower oil, hyssop flower oil, jasmine flower oil,
lavender flower oil, manuka flower oil, Marhoram flower oil, orange
flower oil, rose flower oil, ylang-ylang flower oil, Bark oil,
cassia Bark oil, cinnamon bark oil, sassafras Bark oil, Wood oil,
camphor wood oil, cedar wood oil, rosewood oil, sandalwood oil),
rhizome (ginger) wood oil, resin oil, frankincense oil, myrrh oil,
peel oil, bergamot peel oil, grapefruit peel oil, lemon peel oil,
lime peel oil, orange peel oil, tangerine peel oil, root oil,
valerian oil, Oleic acid, Linoleic acid, Oleyl alcohol, Isostearvl
alcohol, semi-synthetic derivatives thereof, and any combinations
thereof.
[0137] The oil may further comprise a silicone component, such as a
volatile silicone component, which can be the sole oil in the
silicone component or can be combined with other silicone and
non-silicone, volatile and non-volatile oils. Suitable silicone
components include, but are not limited to,
methylphenylpolysiloxane, simethicone, dimethicone,
phenyltrimethicone (or an organomodified version thereof),
alkylated derivatives of polymeric silicones, cetyl dimethicone,
lauryl trimethicone, hydroxylated derivatives of polymeric
silicones, such as dimethiconol, volatile silicone oils, cyclic and
linear silicones, cyclomethicone, derivatives of cyclomethicone,
hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane,
decamethylcyclopentasiloxane, volatile linear
dimethylpolysiloxanes, isohexadecane, isoeicosane, isotetracosane,
polyisobutene, isooctane, isododecane, semi-synthetic derivatives
thereof, and combinations thereof.
[0138] The volatile oil can be the organic solvent, or the volatile
oil can be present in addition to an organic solvent. Suitable
volatile oils include, but are not limited to, a terpene,
monoterpene, sesquiterpene, carminative, azulene, menthol, camphor,
thujone, thymol, nerol, linalool, limonene, geraniol, perillyl
alcohol, nerolidol, famesol, ylangene, bisabolol, farnesene,
ascaridole, chenopodium oil, citronellal, citral, citronellol,
chamazulene, yarrow, guaiazulene, chamomile, semi-synthetic
derivatives, or combinations thereof. In one aspect of the
disclosed, the volatile oil in the silicone component is different
than the oil in the oil phase.
[0139] Surfactants. The surfactant in the nanoemulsion can be a
pharmaceutically acceptable ionic surfactant, a pharmaceutically
acceptable nonionic surfactant, a pharmaceutically acceptable
cationic surfactant, a pharmaceutically acceptable anionic
surfactant, or a pharmaceutically acceptable zwitterionic
surfactant.
[0140] Exemplary useful surfactants are described in Applied
Surfactants: Principles and Applications (Tharwat F. Tadros,
Copyright August 2005 WILEY-VCH Verlag GmbH & Co. KGaA,
Weinheim ISBN: 3-527-30629-3), which is specifically incorporated
by reference. Further, the surfactant can be a pharmaceutically
acceptable ionic polymeric surfactant, a pharmaceutically
acceptable nonionic polymeric surfactant, a pharmaceutically
acceptable cationic polymeric surfactant, a pharmaceutically
acceptable anionic polymeric surfactant, or a pharmaceutically
acceptable zwitterionic polymeric surfactant. Examples of polymeric
surfactants include, but are not limited to, a graft copolymer of a
poly(methyl methacrylate) backbone with multiple (at least one)
polyethylene oxide (PEO) side chain, polyhydroxystearic acid, an
alkoxylated alkyl phenol formaldehyde condensate, a polyalkylene
glycol modified polyester with fatty acid hydrophobes, a polyester,
semi-synthetic derivatives thereof, or combinations thereof.
[0141] Surface active agents or surfactants, are amphipathic
molecules that consist of a nonpolar hydrophobic portion, usually a
straight or branched hydrocarbon or fluorocarbon chain containing
8-18 carbon atoms, attached to a polar or ionic hydrophilic
portion. The hydrophilic portion can be nonionic, ionic or
zwitterionic. The hydrocarbon chain interacts weakly with the water
molecules in an aqueous environment, whereas the polar or ionic
head group interacts strongly with water molecules via dipole or
ion-dipole interactions. Based on the nature of the hydrophilic
group, surfactants are classified into anionic, cationic,
zwitterionic, nonionic and polymeric surfactants.
[0142] Suitable surfactants include, but are not limited to,
ethoxylated nonylphenol comprising 9 to 10 units of ethyleneglycol,
ethoxylated undecanol comprising 8 units of ethyleneglycol,
polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20)
sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate,
polyoxyethylene (20) sorbitan monooleate, sorbitan monolaurate,
sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate,
ethoxylated hydrogenated ricin oils, sodium laurylsulfate, a
diblock copolymer of ethyleneoxyde and propyleneoxyde, Ethylene
Oxide-Propylene Oxide Block Copolymers, and tetra-functional block
copolymers based on ethylene oxide and propylene oxide, Glyceryl
monoesters, Glyceryl caprate, Glyceryl caprylate, Glyceryl cocate,
Glyceryl erucate, Glyceryl hydroxysterate, Glyceryl isostearate,
Glyceryl lanolate, Glyceryl laurate, Glyceryl linolate, Glyceryl
myristate, Glyceryl oleate, Glyceryl PABA, Glyceryl palmitate.
Glyceryl ricinoleate, Glyceryl stearate, Glyceryl thiglycolate,
Glyceryl dilaurate, Glyceryl dioleate, Glyceryl dimyristate,
Glyceryl disterate, Glyceryl sesuioleate, Glyceryl stearate
lactate, Polyoxyethylene cetyl/stearyl ether, Polyoxyethylene
cholesterol ether, Polyoxyethylene laurate or dilaurate,
Polyoxyethylene stearate or distearate, polyoxyethylene fatty
ethers, Polyoxyethylene lauryl ether, Polyoxyethylene stearyl
ether, polyoxyethylene myristyl ether, a steroid, Cholesterol,
Betasitosterol, Bisabolol, fatty acid esters of alcohols, isopropyl
myristate, Aliphati-isopropyl n-butyrate, Isopropyl n-hexanoate,
Isopropyl n-decanoate, Isoproppyl palmitate, Octyldodecyl
myristate, alkoxylated alcohols, alkoxylated acids, alkoxylated
amides, alkoxylated sugar derivatives, alkoxylated derivatives of
natural oils and waxes, polyoxyethylene polyoxypropylene block
copolymers, nonoxynol-14, PEG-8 laurate, PEG-6 Cocoamide, PEG-20
methylglucose sesquistearate, PEG40 lanolin, PEG-40 castor oil,
PEG-40 hydrogenated castor oil, polyoxyethylene fatty ethers,
glyceryl diesters, polyoxyethylene stearyl ether, polyoxyethylene
myristyl ether, and polyoxyethylene lauryl ether, glyceryl
dilaurate, glyceryl dimystate, glyceryl distearate, semi-synthetic
derivatives thereof, or mixtures thereof.
Additional suitable surfactants include, but are not limited to,
non-ionic lipids, such as glyceryl laurate, glyceryl myristate,
glyceryl dilaurate, glyceryl dimyristate, semi-synthetic
derivatives thereof, and mixtures thereof.
[0143] In additional embodiments, the surfactant is a
polyoxyethylene fatty ether having a polyoxyethylene head group
ranging from about 2 to about 100 groups, or an alkoxylated alcohol
having the structure R.sub.5--(OCH.sub.2 CH.sub.2).sub.y--OH,
wherein R.sub.5 is a branched or unbranched alkyl group having from
about 6 to about 22 carbon atoms and y is between about 4 and about
100, and preferably, between about 10 and about 100. Preferably,
the alkoxylated alcohol is the species wherein R.sub.5 is a lauryl
group and y has an average value of 23.
In a different embodiment, the surfactant is an alkoxylated alcohol
which is an ethoxylated derivative of lanolin alcohol. Preferably,
the ethoxylated derivative of lanolin alcohol is laneth-10, which
is the polyethylene glycol ether of lanolin alcohol with an average
ethoxylation value of 10.
[0144] Nonionic surfactants include, but are not limited to, an
ethoxylated surfactant, an alcohol ethoxylated, an alkyl phenol
ethoxylated, a fatty acid ethoxylated, a monoalkaolamide
ethoxylated, a sorbitan ester ethoxylated, a fatty amino
ethoxylated, an ethylene oxide-propylene oxide copolymer,
Bis(polyethylene glycol bis[imidazoyl carbonyl]), nonoxynol-9,
Bis(polyethylene glycol bis[imidazoyl carbonyl]), Brij.RTM. 35,
Brij.RTM. 56, Brij.RTM. 72, Brij.RTM. 76, Brij.RTM. 92V, Brij.RTM.
97, Brij.RTM. 58P, Cremophor.RTM. EL, Decaethylene glycol
monododecyl ether. N-Decanoyl-N-methylglucamine, n-Decyl
alpha-D-glucopyranoside, Decyl beta-D-maltopyranoside,
n-Dodecanoyl-N-methylglucamide, n-Dodecyl alpha-D-maltoside,
n-Dodecyl beta-D-maltoside, n-Dodecyl beta-D-maltoside,
Heptaethylene glycol monodecyl ether, Heptaethylene glycol
monododecyl ether, Heptaethylene glycol monotetradecyl ether,
n-Hexadecyl beta-D-maltoside, Hexaethylene glycol monododecyl
ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol
monooctadecyl ether, Hexaethylene glycol monotetradecyl ether,
Igepal CA-630, Igepal CA-630,
Methyl-6-O-(N-heptylcarbamoyl)-alpha-D-glucopyranoside.
Nonaethylene glycol monododecyl ether,
N-Nonanoyl-N-methylglucamine, N-Nonanoyl-N-methylglucamine,
Octaethylene glycol monodecyl ether, Octaethylene glycol
monododecyl ether, Octaethylene glycol monohexadecyl ether,
Octaethylene glycol monooctadecyl ether, Octaethylene glycol
monotetradecyl ether, Octyl-beta-D-glucopyranoside. Pentaethylene
glycol monodecyl ether, Pentaethylene glycol monododecyl ether,
Pentaethylene glycol monohexadecyl ether, Pentaethylene glycol
monohexyl ether, Pentaethylene glycol monooctadecyl ether,
Pentaethylene glycol monooctyl ether, Polyethylene glycol
diglycidyl ether, Polyethylene glycol ether W-1, Polyoxyethylene 10
tridecyl ether, Polyoxyethylene 100 stearate, Polyoxyethylene 20
isohexadecyl ether, Polyoxyethylene 20 oleyl ether, Polyoxyethylene
40 stearate, Polyoxyethylene 50 stearate, Polyoxyethylene 8
stearate, Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene
25 propylene glycol stearate, Saponin from Quillaja bark, Span.RTM.
20, Span.RTM. 40, Span.RTM. 60, Span.RTM. 65, Span.RTM. 80,
Span.RTM. 85, Tergitol, Type 15-S-12, Tergitol, Type 15-5-30,
Tergitol, Type 15-5-5, Tergitol, Type 15-S-7, Tergitol, Type
15-S-9, Tergitol, Type NP-10, Tergitol. Type NP-4, Tergitol, Type
NP-40, Tergitol, Type NP-7, Tergitol, Type NP-9, Tergitol,
Tergitol, Type TMN-10, Tergitol, Type TMN-6,
Tetradecyl-beta-D-maltoside, Tetraethylene glycol monodecyl ether,
Tetraethylene glycol monododecyl ether, Tetraethylene glycol
monotetradecyl ether, Triethylene glycol monodecyl ether,
Triethylene glycol monododecyl ether, Triethylene glycol
monohexadecyl ether, Triethylene glycol monooctyl ether,
Triethylene glycol monotetradecyl ether, Triton CF-21, Triton
CF-32, Triton DF-12, Triton DF-16, Triton GR-5M, Triton QS-15,
Triton QS-44, Triton X-100, Triton X-102, Triton X-15, Triton
X-151, Triton X-200, Triton X-207, Triton.RTM. X-100, Triton.RTM.
X-114, Triton.RTM. X-165, Triton.RTM. X-305, Triton.RTM. X-405,
Triton.RTM. X-45, Triton.RTM. X-705-70, TWEEN.RTM. 20, TWEEN.RTM.
21, TWEEN.RTM. 40, TWEEN.RTM. 60, TWEEN.RTM. 61, TWEEN.RTM. 65,
TWEEN.RTM. 80, TWEEN.RTM. 81, TWEEN.RTM. 85, Tyloxapol, n-Undecyl
beta-D-glucopyranoside, semi-synthetic derivatives thereof, or
combinations thereof.
[0145] In addition, the nonionic surfactant can be a poloxamer.
Poloxamers are polymers made of a block of polyoxyethylene,
followed by a block of polyoxypropylene, followed by a block of
polyoxyethylene. The average number of units of polyoxyethylene and
polyoxypropylene varies based on the number associated with the
polymer. For example, the smallest polymer, Poloxamer 101, consists
of a block with an average of 2 units of polyoxyethylene, a block
with an average of 16 units of polyoxypropylene, followed by a
block with an average of 2 units of polyoxyethylene. Poloxamers
range from colorless liquids and pastes to white solids. In
cosmetics and personal care products, Poloxamers are used in the
formulation of skin cleansers, bath products, shampoos, hair
conditioners, mouthwashes, eye makeup remover and other skin and
hair products. Examples of Poloxamers include, but are not limited
to, Poloxamer 101, Poloxamer 105, Poloxamer 108, Poloxamer 122,
Poloxamer 123, Poloxamer 124, Poloxamer 181, Poloxamer 182,
Poloxamer 183, Poloxamer 184, Poloxamer 185, Poloxamer 188,
Poloxamer 212. Poloxamer 215, Poloxamer 217, Poloxamer 231,
Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238,
Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 331,
Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338.
Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer 407,
Poloxamer 105 Benzoate, and Poloxamer 182 Dibenzoate.
[0146] Suitable cationic surfactants include, but are not limited
to, a quarternary ammonium compound, an alkyl trimethyl ammonium
chloride compound, a dialkyl dimethyl ammonium chloride compound, a
cationic halogen-containing compound, such as cetylpyridinium
chloride, Benzalkonium chloride, Benzalkonium chloride,
Benzyldimethylhexadecylammonium chloride,
Benzyldimethyltetradecylammonium chloride,
Benzyldodecyldimethylammonium bromide, Benzyltrimethylammonium
tetrachloroiodate, Dimethyldioctadecylammonium bromide,
Dodecylethyldimethylammonium bromide, Dodecyltrimethylammonium
bromide, Dodecyltrimethylammonium bromide,
Ethylhexadecyldimethylammonium bromide, Girard's reagent T,
Hexadecyltrimethylammonium bromide, Hexadecyltrimethylammonium
bromide, N,N',N'-Polyoxyethylene(10)-N-tallow-1,3-diaminopropane,
Thonzonium bromide, Trimethyl(tetradecyl)ammonium bromide,
1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, 1-Decanaminium,
N-decyl-N, N-dimethyl-, chloride, Didecyl dimethyl ammonium
chloride, 2-(2-(p-(Diisobutyl)cresosxy)ethoxy)ethyl dimethyl benzyl
ammonium chloride, 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl
dimethyl benzyl ammonium chloride, Alkyl 1 or 3
benzyl-1-(2-hydroxethyl)-2-imidazolinium chloride, Alkyl
bis(2-hydroxyethyl) benzyl ammonium chloride, Alkyl demethyl benzyl
ammonium chloride, Alkyl dimethyl 3,4-dichlorobenzyl ammonium
chloride (100% C.sub.12), Alkyl dimethyl 3,4-dichlorobenzyl
ammonium chloride (50% C.sub.14, 40% C.sub.12, 10% C.sub.16), Alkyl
dimethyl 3,4-dichlorobenzyl ammonium chloride (55% C.sub.14, 23%
C.sub.12. 20% C.sub.16). Alkyl dimethyl benzyl ammonium chloride,
Alkyl dimethyl benzyl ammonium chloride (100% C.sub.14), Alkyl
dimethyl benzyl ammonium chloride (100% C.sub.16), Alkyl dimethyl
benzyl ammonium chloride (41% C.sub.14, 28% C.sub.12), Alkyl
dimethyl benzyl ammonium chloride (47% C.sub.12, 18% C.sub.14),
Alkyl dimethyl benzyl ammonium chloride (55% C16, 20% C.sub.14),
Alkyl dimethyl benzyl ammonium chloride (58% C.sub.14, 28%
C.sub.16), Alkyl dimethyl benzyl ammonium chloride (60% C.sub.14,
25% C.sub.12), Alkyl dimethyl benzyl ammonium chloride (61%
C.sub.11, 23% C.sub.14), Alkyl dimethyl benzyl ammonium chloride
(61% C.sub.12, 23% C.sub.14), Alkyl dimethyl benzyl ammonium
chloride (65% C.sub.12, 25% C.sub.14), Alkyl dimethyl benzyl
ammonium chloride (67% C.sub.12, 24% C.sub.14), Alkyl dimethyl
benzyl ammonium chloride (67% C.sub.12, 25% C.sub.14), Alkyl
dimethyl benzyl ammonium chloride (90% C.sub.14, 5% C.sub.12),
Alkyl dimethyl benzyl ammonium chloride (93% C.sub.14, 4%
C.sub.12), Alkyl dimethyl benzyl ammonium chloride (95% C.sub.16,
5% C.sub.18), Alkyl dimethyl benzyl ammonium chloride, Alkyl
didecyl dimethyl ammonium chloride, Alkyl dimethyl benzyl ammonium
chloride, Alkyl dimethyl benzyl ammonium chloride (C.sub.12-16).
Alkyl dimethyl benzyl ammonium chloride (C.sub.12-18), Alkyl
dimethyl benzyl ammonium chloride, dialkyl dimethyl benzyl ammonium
chloride, Alkyl dimethyl dimethybenzyl ammonium chloride, Alkyl
dimethyl ethyl ammonium bromide (90% C.sub.14, 5% C.sub.16, 5%
C.sub.12), Alkyl dimethyl ethyl ammonium bromide (mixed alkyl and
alkenyl groups as in the fatty acids of soybean oil), Alkyl
dimethyl ethylbenzyl ammonium chloride, Alkyl dimethyl ethylbenzyl
ammonium chloride (60% C.sub.14), Alkyl dimethyl isopropylbenzyl
ammonium chloride (50% C.sub.12, 30% C.sub.14, 17% C.sub.16, 3%
C.sub.18), Alkyl trimethyl ammonium chloride (58% C.sub.18, 40%
C.sub.16, 1% C.sub.14, 1% C.sub.12), Alkyl trimethyl ammonium
chloride (90% C.sub.18, 10% C.sub.16), Alkyldimethyl(ethylbenzyl)
ammonium chloride (C.sub.12-18), Di-(C.sub.8-10)-alkyl dimethyl
ammonium chlorides, Dialkyl dimethyl ammonium chloride, Dialkyl
methyl benzyl ammonium chloride, Didecyl dimethyl ammonium
chloride, Diisodecyl dimethyl ammonium chloride, Dioctyl dimethyl
ammonium chloride, Dodecyl bis (2-hydroxyethyl) octyl hydrogen
ammonium chloride, Dodecyl dimethyl benzyl ammonium chloride,
Dodecylcarbamoyl methyl dimethyl benzyl ammonium chloride,
Heptadecyl hydroxyethylimidazolinium chloride.
Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine,
Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, Myristalkonium
chloride (and) Quat RNIUM 14, N,N-Dimethyl-2-hydroxypropylammonium
chloride polymer, n-Tetradecyl dimethyl benzyl ammonium chloride
monohydrate, Octyl decyl dimethyl ammonium chloride, Octyl dodecyl
dimethyl ammonium chloride, Octyphenoxyethoxyethyl dimethyl benzyl
ammonium chloride, Oxydiethylenebis(alkyl dimethyl ammonium
chloride), Quaternary ammonium compounds, dicoco alkyldimethyl,
chloride, Trimethoxysily propyl dimethyl octadecyl ammonium
chloride. Trimethoxysilyl quats, Trimethyl dodecylbenzyl ammonium
chloride, semi-synthetic derivatives thereof, and combinations
thereof.
[0147] Exemplary cationic halogen-containing compounds include, but
are not limited to, cetylpyridinium halides, cetyltrimethylammonium
halides, cetyldimethylethylammonium halides,
cetyldimethylbenzylammonium halides, cetyltributylphosphonium
halides, dodecyltrimethylammonium halides, or
tetradecyltrimethylammonium halides. In some particular
embodiments, suitable cationic halogen containing compounds
comprise, but are not limited to, cetylpyridinium chloride (CPC),
cetyltrimethylammonium chloride, cetylbenzyldimethylammonium
chloride, cetylpyridinium bromide (CPB), cetyltrimethylammonium
bromide (CTAB), cetyidimethylethylammonium bromide,
cetyltributylphosphonium bromide, dodecyltrimethylammonium bromide,
and tetrad ecyltrimethylammonium bromide. In particularly preferred
embodiments, the cationic halogen containing compound is CPC,
although the compositions of the present disclosed are not limited
to formulation with an particular cationic containing compound.
[0148] Suitable anionic surfactants include, but are not limited
to, a carboxylate, a sulphate, a sulphonate, a phosphate,
chenodeoxycholic acid, chenodeoxycholic acid sodium salt, cholic
acid, ox or sheep bile, Dehydrocholic acid, Deoxycholic acid,
Deoxycholic acid, Deoxycholic acid methyl ester, Digitonin.
Digitoxigenin, N,N-Dimethyldodecylamine N-oxide, Docusate sodium
salt, Glycochenodeoxycholic acid sodium salt, Glycocholic acid
hydrate, synthetic, Glycocholic acid sodium salt hydrate,
synthetic, Glycodeoxycholic acid monohydrate, Glycodeoxycholic acid
sodium salt, Glycodeoxycholic acid sodium salt, Glycolithocholic
acid 3-sulfate disodium salt, Glycolithocholic acid ethyl ester,
N-Lauroylsarcosine sodium salt, N-Lauroylsarcosine solution,
N-Lauroylsarcosine solution, Lithium dodecyl sulfate, Lithium
dodecyl sulfate, Lithium dodecyl sulfate, Lugol solution, Niaproof
4, Type 4, 1-Octanesulfonic acid sodium salt, Sodium
1-butanesulfonate, Sodium 1-decanesulfonate, Sodium
1-decanesulfonate, Sodium 1-dodecanesulfonate, Sodium
1-heptanesulfonate anhydrous, Sodium 1-heptanesulfonate anhydrous,
Sodium 1-nonanesulfonate, Sodium 1-propanesulfonate monohydrate,
Sodium 2-bromoethanesulfonate, Sodium cholate hydrate, Sodium
choleate, Sodium deoxycholate, Sodium deoxycholate monohydrate.
Sodium dodecyl sulfate, Sodium hexanesulfonate anhydrous, Sodium
octyl sulfate, Sodium pentanesulfonate anhydrous, Sodium
taurocholate, Taurochenodeoxycholic acid sodium salt,
Taurodeowcholic acid sodium salt monohydrate, Taurohyodeoxycholic
acid sodium salt hydrate, Taurolithocholic acid 3-sulfate disodium
salt, Tauroursodeoxycholic acid sodium salt, Trizma.RTM. dodecyl
sulfate, TWEEN.RTM. 80, Ursodeoxycholic acid, semi-synthetic
derivatives thereof, and combinations thereof.
Suitable zwitterionic surfactants include, but are not limited to,
an N-alkyl betaine, lauryl amindo propyl dimethyl betaine, an alkyl
dimethyl glycinate, an N-alkyl amino propionate, CHAPS, minimum 98%
(TLC), CHAPS, SigmaUltra, minimum 98% (TLC), CHAPS, for
electrophoresis, minimum 98% (TLC), CHAPSO, minimum 98%, CHAPSO,
SigmaUltra, CHAPSO, for electrophoresis,
3-(Decyldimethylammonio)propanesulfonate inner salt,
3-Dodecyldimethylammonio)propanesulfonate inner salt, SigmaUltra,
3-(Dodecyldimethylammonio)propanesulfonate inner salt,
3-(N,N-Dimethylmyristylammonio)propanesulfonate,
3-(N,N-Dimethyloctadecylammonio)propanesulfonate,
3-(N,N-Dimethyloctylammonio)propanesulfonate inner salt,
3-(N,N-Dimethylpalmitylammonio)propanesulfonate, semi-synthetic
derivatives thereof, and combinations thereof.
[0149] In some embodiments, the nanoemulsion comprises a cationic
surfactant, which can be cetylpyridinium chloride. In other
embodiments of the disclosed, the nanoemulsion comprises a cationic
surfactant, and the concentration of the cationic surfactant is
less than about 5.0% and greater than about 0.001%. In yet another
embodiment of the disclosed, the nanoemulsion comprises a cationic
surfactant, and the concentration of the cationic surfactant is
selected from the group consisting of less than about 5%, less than
about 4.5%, less than about 4.0%, less than about 3.5%, less than
about 3.0%, less than about 2.5%, less than about 2.0%, less than
about 1.5%, less than about 1.0%, less than about 0.90%, less than
about 0.80%, less than about 0.70%, less than about 0.60%, less
than about 0.50%, less than about 0.40%, less than about 0.30%,
less than about 0.20%, or less than about 0.10%. Further, the
concentration of the cationic agent in the nanoemulsion is greater
than about 0.002%, greater than about 0.003%, greater than about
0.004%, greater than about 0.005%, greater than about 0.006%,
greater than about 0.007%, greater than about 0.008%, greater than
about 0.009%, greater than about 0.010%, or greater than about
0.001%. In one embodiment, the concentration of the cationic agent
in the nanoemulsion is less than about 5.0% and greater than about
0.001%.
[0150] In another embodiment, the nanoemulsion comprises at least
one cationic surfactant and at least one non-cationic surfactant.
The non-cationic surfactant is a nonionic surfactant, such as a
polysorbate (Tween), such as polysorbate 80 or polysorbate 20. In
one embodiment, the non-ionic surfactant is present in a
concentration of about 0.01% to about 5.0%, or the non-ionic
surfactant is present in a concentration of about 0.1% to about 3%.
In yet another embodiment, the nanoemulsion comprises a cationic
surfactant present in a concentration of about 0.01% to about 2%,
in combination with a nonionic surfactant.
[0151] Additional Ingredients. Additional compounds suitable for
use in the nanoemulsion include but are not limited to one or more
solvents, such as an organic phosphate-based solvent, bulking
agents, coloring agents, pharmaceutically acceptable excipients, a
preservative, pH adjuster, buffer, chelating agent, etc. The
additional compounds can be admixed into a previously emulsified
nanoemulsion vaccine, or the additional compounds can be added to
the original mixture to be emulsified. In certain of these
embodiments, one or more additional compounds are admixed into an
existing nanoemulsion composition immediately prior to its use.
[0152] Suitable preservatives in the nanoemulsion include, but are
not limited to, cetylpyridinium chloride, benzalkonium chloride,
benzyl alcohol, chlorhexidine, imidazolidinyl urea, phenol,
potassium sorbate, benzoic acid, bronopol, chlorocresol, paraben
esters, phenoxyethanol, sorbic acid, alpha-tocophernol, ascorbic
acid, ascorbyl palmitate, butylated hydroxyanisole, butylated
hydroxytoluene, sodium ascorbate, sodium metabisulphite, citric
acid, edetic acid, semi-synthetic derivatives thereof, and
combinations thereof. Other suitable preservatives include, but are
not limited to, benzyl alcohol, chlorhexidine (bis
(p-chlorophenyldiguanido) hexane), chlorphenesin
(3-(-4-chloropheoxy)-propane-1,2-diol), Kathon CG (methyl and
methylchloroisothiazolinone), parabens (methyl, ethyl, propyl,
butyl hydrobenzoates), phenoxyethanol (2-phenoxyethanol), sorbic
acid (potassium sorbate, sorbic acid), Phenonip (phenoxyethanol,
methyl, ethyl, butyl, propyl parabens), Phenoroc (phenoxyethanol
0.73%, methyl paraben 0.2%, propyl paraben 0.07%), Liquipar Oil
(isopropyl, isobutyl, butylparabens), Liquipar PE (70%
phenoxyethanol, 30% liquipar oil), Nipaguard MPA (benzyl alcohol
(70%), methyl & propyl parabens), Nipaguard MPS (propylene
glycol, methyl & propyl parabens), Nipasept (methyl, ethyl and
propyl parabens), Nipastat (methyl, butyl, ethyl and propyel
parabens), Elestab 388 (phenoxyethanol in propylene glycol plus
chlorphenesin and methylparaben), and Killitol (7.5% chlorphenesin
and 7.5% methyl parabens).
The nanoemulsion vaccine may further comprise at least one pH
adjuster. Suitable pH adjusters in the nanoemulsion vaccine of the
disclosed include, but are not limited to, diethyanolamine, lactic
acid, monoethanolamine, triethylanolamine, sodium hydroxide, sodium
phosphate, semi-synthetic derivatives thereof, and combinations
thereof.
[0153] In addition, the nanoemulsion can comprise a chelating
agent. In one embodiment, the chelating agent is present in an
amount of about 0.0005% to about 1%. Examples of chelating agents
include, but are not limited to, ethylenediamine,
ethylenediaminetetraacetic acid (EDTA), phytic acid, polyphosphoric
acid, citric acid, gluconic acid, acetic acid, lactic acid, and
dimercaprol, and a preferred chelating agent is
ethylenediaminetetraacetic acid. The nanoemulsion can comprise a
buffering agent, such as a pharmaceutically acceptable buffering
agent. Examples of buffering agents include, but are not limited
to, 2-Amino-2-methyl-1,3-propanediol, .gtoreq.99.5% (NT),
2-Amino-2-methyl-1-propanol, .gtoreq.99.0% (GC), L-(+)-Tartaric
acid, .gtoreq.99.5% (T), ACES, .gtoreq.99.5% (T), ADA,
.gtoreq.99.0% (T), Acetic acid, .gtoreq.99.5% (GC/T), Acetic acid,
for luminescence, .gtoreq.99.5% (GC/T), Ammonium acetate solution,
for molecular biology, .about.5 M in H.sub.2O, Ammonium acetate,
for luminescence, .gtoreq.99.0% (calc. on dry substance, T),
Ammonium bicarbonate, .gtoreq.99.5% (T), Ammonium citrate dibasic,
.gtoreq.99.0% (T), Ammonium formate solution, 10 M in H.sub.2O,
Ammonium formate, 99.0% (calc. based on dry substance, NT),
Ammonium oxalate monohydrate, .gtoreq.99.5% (RT), Ammonium
phosphate dibasic solution, 2.5 M in H.sub.2O, Ammonium phosphate
dibasic, 99.0% (T), Ammonium phosphate monobasic solution, 2.5 M in
H.sub.2O, Ammonium phosphate monobasic..gtoreq.99.5% (T), Ammonium
sodium phosphate dibasic tetrahydrate, .gtoreq.99.5% (NT), Ammonium
sulfate solution, for molecular biology, 3.2 M in H.sub.2O,
Ammonium tartrate dibasic solution. 2 M in H.sub.2O (colorless
solution at 20.degree. C.), Ammonium tartrate dibasic. 99.5% (T),
BES buffered saline, for molecular biology, 2.times. concentrate,
BES, .gtoreq.99.5% (T), BES, for molecular biology, .gtoreq.99.5%
(T), BICINE buffer Solution, for molecular biology, 1 M in
H.sub.2O, BICINE. 99.5% (T), BIS-TRIS, .gtoreq.99.0% (NT),
Bicarbonate buffer solution, >0.1 M Na.sub.2CO.sub.3, >0.2 M
NaHHCO.sub.3, Boric acid, .gtoreq.99.5% (T), Boric acid, for
molecular biology, .gtoreq.99.5% (T), CAPS..gtoreq.99.0% (TLC),
CHES, .gtoreq.99.5% (T), Calcium acetate hydrate, .gtoreq.99.0%
(calc. on dried material, KT). Calcium carbonate, precipitated,
.gtoreq.99.0% (KT), Calcium citrate tribasic tetrahydrate. 98.0%
(calc. on dry substance, KT), Citrate Concentrated Solution, for
molecular biology, 1 M in H.sub.2O, Citric acid, anhydrous, 99.5%
(T), Citric acid, for luminescence, anhydrous, .gtoreq.99.5% (T),
Diethanolamine, .gtoreq.99.5% (GC), EPPS, .gtoreq.99.0% (T),
Ethylenediaminetetraacetic acid disodium salt dihydrate, for
molecular biology, .gtoreq.99.0% (T), Formic acid solution, 1.0 M
in H.sub.2O, Gly-Gly-Gly, .gtoreq.99.0% (NT), Gly-Gly,
.gtoreq.99.5% (NT), Glycine, .gtoreq.99.0% (NT), Glycine, for
luminescence, .gtoreq.99.00% (NT), Glycine, for molecular biology,
.gtoreq.99.0% (NT), HEPES buffered saline, for molecular biology,
2.times. concentrate, HEPES, .gtoreq.99.5% (T), HEPES, for
molecular biology, .gtoreq.99.5% (T), Imidazole buffer Solution. 1
M in H.sub.2O, Imidazole, .gtoreq.99.5% (GC), Imidazole, for
luminescence, .gtoreq.99.5% (GC), Imidazole, for molecular biology,
.gtoreq.99.5% (GC), Lipoprotein Refolding Buffer. Lithium acetate
dihydrate, .gtoreq.99.0% (NT), Lithium citrate tribasic
tetrahydrate, .gtoreq.99.5% (NT), MES hydrate, .gtoreq.99.5% (T),
MES monohydrate, for luminescence, .gtoreq.99.5% (T), MES solution,
for molecular biology, 0.5 M in H.sub.2O, MOPS, .gtoreq.99.5% (T),
MOPS, for luminescence, .gtoreq.99.5% (T), MOPS, for molecular
biology, .gtoreq.99.5% (T), Magnesium acetate solution, for
molecular biology, .about.1 M in H.sub.2O. Magnesium acetate
tetrahydrate, .gtoreq.99.0% (KT), Magnesium citrate tribasic
nonahydrate, .gtoreq.98.0% (calc. based on dry substance, KT),
Magnesium formate solution, 0.5 M in H.sub.2O, Magnesium phosphate
dibasic trihydrate, .gtoreq.98.0% (KT), Neutralization solution for
the in-situ hybridization for in-situ hybridization, for molecular
biology, Oxalic acid dihydrate, .gtoreq.99.5% (RT), PIPES,
.gtoreq.99.5% (T), PIPES, for molecular biology, .gtoreq.99.5% (T),
Phosphate buffered saline, solution (autoclaved), Phosphate
buffered saline washing buffer for peroxidase conjugates in Western
Blotting, 10.times. concentrate, Piperazine, anhydrous,
.gtoreq.99.0% (T), Potassium D-tartrate monobasic, .gtoreq.99.0%
(T), Potassium acetate solution, for molecular biology, Potassium
acetate solution, for molecular biology, 5 M in H.sub.2O, Potassium
acetate solution, for molecular biology, .about.1 M in H.sub.2O,
Potassium acetate, .gtoreq.99.0% (NT), Potassium acetate, for
luminescence, .gtoreq.99.0% (NT), Potassium acetate, for molecular
biology, .gtoreq.99.0% (NT), Potassium bicarbonate, .gtoreq.99.5%
(T), Potassium carbonate, anhydrous, .gtoreq.99.0% (T), Potassium
chloride, .gtoreq.99.5% (AT), Potassium citrate monobasic,
.gtoreq.99.0% (dried material, NT), Potassium citrate tribasic
solution. 1 M in H.sub.2O, Potassium formate solution, 14 M in
H.sub.2O, Potassium formate, .gtoreq.99.5% (NT), Potassium oxalate
monohydrate, .gtoreq.99.0% (RT), Potassium phosphate dibasic,
anhydrous, .gtoreq.99.0% (T), Potassium phosphate dibasic, for
luminescence, anhydrous, .gtoreq.99.0% (T), Potassium phosphate
dibasic, for molecular biology, anhydrous, .gtoreq.99.0% (T),
Potassium phosphate monobasic, anhydrous, .gtoreq.99.5% (T),
Potassium phosphate monobasic, for molecular biology, anhydrous,
.gtoreq.99.5% (T), Potassium phosphate tribasic monohydrate,
.gtoreq.95% (T), Potassium phthalate monobasic, .gtoreq.99.5% (T),
Potassium sodium tartrate solution, 1.5 M in H.sub.2O, Potassium
sodium tartrate tetrahydrate, .gtoreq.99.5% (NT), Potassium
tetraborate tetrahydrate, .gtoreq.99.0% (T), Potassium tetraoxalate
dihydrate, .gtoreq.99.5% (RT), Propionic acid solution, 1.0 M in
H.sub.2O, STE buffer solution, for molecular biology, pH 7.8, STET
buffer solution, for molecular biology, pH 8.0. Sodium
5,5-diethylbarbiturate, .gtoreq.99.5% (NT), Sodium acetate
solution, for molecular biology, .about.3 M in H.sub.2O, Sodium
acetate trihydrate, .gtoreq.99.5% (NT), Sodium acetate, anhydrous,
.gtoreq.99.0% (NT), Sodium acetate for luminescence, anhydrous,
.gtoreq.99.0% (NT), Sodium acetate, for molecular biology,
anhydrous, .gtoreq.99.0% (NT), Sodium bicarbonate, .gtoreq.99.5%
(T). Sodium bitartrate monohydrate, .gtoreq.99.0% (T), Sodium
carbonate decahydrate, .gtoreq.99.5% (T), Sodium carbonate,
anhydrous, .gtoreq.99.5% (calc. on dry substance, T), Sodium
citrate monobasic, anhydrous, .gtoreq.99.5% (T), Sodium citrate
tribasic dihydrate, .gtoreq.99.0% (NT), Sodium citrate tribasic
dihydrate, for luminescence, .gtoreq.99.0% (NT), Sodium citrate
tribasic dihydrate, for molecular biology, .gtoreq.99.5% (NT),
Sodium formate solution, 8 M in H.sub.2O, Sodium oxalate,
.gtoreq.99.5% (RT), Sodium phosphate dibasic dihydrate,
.gtoreq.99.0% (T), Sodium phosphate dibasic dihydrate, for
luminescence, .gtoreq.99.0% (T), Sodium phosphate dibasic
dihydrate, for molecular biology, .gtoreq.99.0% (T), Sodium
phosphate dibasic dodecahydrate, .gtoreq.99.0% (T), Sodium
phosphate dibasic solution, 0.5 M in H.sub.2O, Sodium phosphate
dibasic, anhydrous, .gtoreq.99.5% (T), Sodium phosphate dibasic,
for molecular biology, .gtoreq.99.5% (T). Sodium phosphate
monobasic dihydrate, .gtoreq.99.0% (T), Sodium phosphate monobasic
dihydrate, for molecular biology, .gtoreq.99.0% (T), Sodium
phosphate monobasic monohydrate, for molecular biology,
.gtoreq.99.5% (T), Sodium phosphate monobasic solution. 5 M in
H.sub.2O, Sodium pyrophosphate dibasic, .gtoreq.99.0% (T), Sodium
pyrophosphate tetrabasic decahydrate, .gtoreq.99.5% (T), Sodium
tartrate dibasic dihydrate, .gtoreq.99.0% (NT), Sodium tartrate
dibasic solution, 1.5 M in H.sub.2O (colorless solution at
20.degree. C.), Sodium tetraborate decahydrate, .gtoreq.99.5% (T),
TAPS, .gtoreq.99.5% (T), TES, .gtoreq.99.5% (calc. based on dry
substance, T), TM buffer solution, for molecular biology, pH 7.4.
TNT buffer solution, for molecular biology, pH 8.0, TRIS Glycine
buffer solution, 10.times. concentrate, TRIS acetate-EDTA buffer
solution, for molecular biology, TRIS buffered saline, 10.times.
concentrate, TRIS glycine SDS buffer solution, for electrophoresis,
10.times. concentrate, TRIS phosphate-EDTA buffer solution, for
molecular biology, concentrate, 10.times. concentrate, Tricine,
.gtoreq.99.5% (NT), Triethanolamine, .gtoreq.99.5% (GC),
Triethylamine, .gtoreq.99.5% (GC), Triethylammonium acetate buffer,
volatile buffer, .about.1.0 M in H.sub.2O, Triethylammonium
phosphate solution, volatile buffer, .about.1.0 M in H.sub.2O,
Trimethylammonium acetate solution, volatile buffer, .about.1.0 M
in H.sub.2O, Trimethylammonium phosphate solution, volatile buffer,
.about.1 M in H.sub.2O, Tris-EDTA buffer solution, for molecular
biology, concentrate, 100.times. concentrate, Tris-EDTA buffer
solution, for molecular biology, pH 7.4, Tris-EDTA buffer solution,
for molecular biology, pH 8.0, Trizma.RTM. acetate, .gtoreq.99.0%
(NT), Trizma.RTM. base, .gtoreq.99.8% (T), Trizma.RTM. base,
.gtoreq.99.8% (T), Trizma.RTM. base, for luminescence,
.gtoreq.99.8% (T), Trizma.RTM. base, for molecular biology,
.gtoreq.99.8% (T), Trizma.RTM. carbonate, .gtoreq.98.5% (T),
Trizma.RTM. hydrochloride buffer solution, for molecular biology,
pH 7.2, Trizma.RTM. hydrochloride buffer solution, for molecular
biology, pH 7.4, Trizma.RTM. hydrochloride buffer solution, for
molecular biology, pH 7.6, Trizma.RTM. hydrochloride buffer
solution, for molecular biology, pH 8.0, Trizma.RTM. hydrochloride,
.gtoreq.99.0% (AT), Trizma.RTM. hydrochloride, for luminescence,
.gtoreq.99.0% (AT), Trizma.RTM. hydrochloride for molecular
biology, .gtoreq.99.0% (AT), and Trizma.RTM. maleate, .gtoreq.99.5%
(NT).
[0154] The nanoemulsion can comprise one or more emulsifying agents
to aid in the formation of emulsions. Emulsifying agents include
compounds that aggregate at the oil/water interface to form a kind
of continuous membrane that prevents direct contact between two
adjacent droplets. Certain embodiments feature nanoemulsion that
may readily be diluted with water or another aqueous phase to a
desired concentration without impairing their desired
properties.
[0155] As described herein, an immunogenic composition of the
invention may comprise one or more immunostimulatory compounds.
Examples of immunostimulatory compounds include, but are not
limited to, chitosan, glucan, enterotoxin, nucleic acid (e.g., CpG
motifs), MF59, alum, ASO system, etc. Indeed, a variety of
immunostimulatory compounds known in the art may be used in an
adjuvant formulation (e.g., for use in an immunogenic composition)
of the invention. Immunostimulatory compounds include, but are not
limited to, a TLR antagonist (e.g., a polyinosinic-polycytidylic
acid (poly (IC)) (e.g., that activates TLR3); Pam3CSK4 (e.g., that
activates TLR1/2); FSL-1 and/or MALP2 (e.g., that activate TLR2/6);
monophosphoryl lipid A, (MPL) (e.g., that activate TLR4); flagellin
(e.g., that activate TLR5); imiquimod (e.g., that activate TLR7); a
Class B CpG phosphorothioate oligodeoxynucleotide, (CpG ODN) (e.g.,
that activate TLR9); a multi-pattern recognition receptors
(multi-PRR ligand) (e.g., that act as agonists for dual or multiple
TLR receptors and other PRRs)); a RIG-I-like receptor agonist
(e.g., 5'ppp dsRNA and/or Poly(I:C)/LyoVec complexes); NOD and
NOD-like receptor ligand agonists; inflammasome inducers (e.g.,
alum, alum salts, and/or alum crystals, chitosan, etc.); cytosolic
DNA Sensors (CDS) and STING Ligands; immune cell receptors (e.g.,
CD40L, FMS-like tyrosine kinase ligand (e.g., Flt3 ligand), and
multimeric immune cell receptors): Cytokines and chemokines (e.g.,
IL-1, IL-6, TGF-.beta., IL-12, IL-15, interferons (.alpha., .beta.,
.gamma., .delta.), GM-CSF and others); synthetic glycolipids (e.g.,
alpha-galactosylceramide); and/or vitamins (e.g., retinoic acid).
It is within the purview of one of ordinary skill in the art to
employ suitable immunostimulatory compounds/immune modulators in
the context of the present disclosure
[0156] An immunostimulatory compound can be present in an
immunogenic composition at any pharmaceutically acceptable amount
including, but not limited to, from about 0.001% up to about 10%,
and any amount in between, such as about 0.01%, about 0.02%, about
0.03%. about 0.04%, about 0.05%, about 0.06%, about 0.07%, about
0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%,
about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about
1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,
about 8%, about 9%, or about 10%.
[0157] Exemplary Nanoemulsions. An exemplary nanoemulsion adjuvant
composition according to the invention is designated "W805EC" or
"DODAC NE" adjuvant. The composition of W805EC or DODAC NE adjuvant
is shown in Table 1. The mean droplet size for the either adjuvant
is .about.400-500 nm. All of the components of the nanoemulsion are
included on the FDA inactive ingredient list for Approved Drug
Products.
TABLE-US-00001 TABLE 1 Exemplary Nanoemulsion Formulations.
Function Nanoemulsion-Adjuvant Aqueous Diluent Purified Water, USP
Hydrophobic Oil (Core) Soybean Oil, USP (super refined) Organic
Solvent Dehydrated Alcohol, USP (anhydrous ethanol) Surfactant
Polysorbate 80, NF Emulsifying Agent Cetylpyridinium Chloride
(CPC), Preservative USP or Dioctadecyldimethylammonium chloride
(DODAC)
[0158] The nanoemulsion adjuvants may be formed by emulsification
of an oil, purified water, nonionic detergent, organic solvent and
surfactant, such as a cationic surfactant. An exemplary specific
nanoemulsion adjuvant is designated as "60% W805EC" or 60% DODAC
NE". The 60% W805EC-vaccine adjuvant or 60% DODAC-vaccine adjuvant
is composed of the ingredients shown in Table 2, below: purified
water. USP; soybean oil USP; Dehydrated Alcohol, USP [anhydrous
ethanol]; Polysorbate 80, NF, cetylpyridinium chloride, USP (CPC)
and/or dioctadecyldimethylammonium chloride (DODAC). All components
of this exemplary nanoemulsion adjuvant are included on the FDA
list of approved inactive ingredients for Approved Drug
Products.
TABLE-US-00002 TABLE 2 Composition of 60% W805EC-Adjuvant (w/w %).
60% 60% Ingredients W.sub.80SEC DODAC NE Purified Water, USP 54.10%
54.10% Soybean Oil, USP 37.67% 37.67% Dehydrated Alcohol, USP 4.04%
4.04% (anhydrous ethanol) Polysorbate 80, NF 3.55% 3.55%
Cetylpyridinium Chloride (CPC), USP 0.64% --
Dioctadecyldimethylammonium -- 0.64% chloride (DODAC)
For the purposes of the present disclosure, a nanoemulsion as
provided here (e.g. W805EC or DODAC NE) can make up between 1-99%
(w/w %) of an immunogenic composition (e.g., a vaccine composition)
of the disclosure. For instance, the nanoemulsion can be about 1,
about 5, about 10, about 15, about 20, about 25, about 30, about
35, about 40, about 45, about 50, about 55, about 60, about 65,
about 70, about 75, about 80, about 85, about 90, about 95, or
about 99% of a vaccine formulation of the disclosure.
[0159] Pharmaceutical Compositions. Immunogenic compositions of the
present disclosure may be formulated into pharmaceutical
compositions, such as a vaccine, that are administered in a
therapeutically effective amount to a subject and may further
comprise suitable, pharmaceutically-acceptable excipients,
additives, or preservatives. Suitable excipients, additives, and
preservatives are well known in the art.
[0160] By the phrase "therapeutically effective amount" it is meant
any amount of the composition that is effective in preventing,
treating, or ameliorating a disease, pathogen, malignancy, or
condition associated with the protein or antigen present in
immunogenic composition. By "protective immune response" it is
meant that the immune response is associated with prevention,
treating, or amelioration of a disease. Complete prevention is not
required, though is encompassed by the present disclosure. The
immune response can be evaluated using the methods discussed herein
or by any method known by a person of skill in the art.
[0161] The pharmaceutical compositions may be formulated for
immediate release, sustained release, controlled release, delayed
release, or any combinations thereof, into the epidermis or dermis.
In some embodiments, the formulations may comprise a
penetration-enhancing agent. Suitable penetration-enhancing agents
include, but are not limited to, alcohols such as ethanol,
triglycerides and aloe compositions. The amount of the
penetration-enhancing agent may comprise from about 0.5% to about
40% by weight of the formulation.
[0162] In one aspect of the disclosure, the invention relates to a
method for vaccination against, or for prophylaxis or therapy
(prevention or treatment) of exposure to, infection with, or
poisoning by a pathogen (e.g., a bacterial, viral, and/or fungal
pathogen) via administration of a therapeutically or
prophylactically effective amount of (a pharmaceutical composition
comprising) an immunogenic composition of the disclosure as defined
above, or obtainable as defined herein, to a subject in need of
prophylaxis or therapy.
[0163] An immunogenic compositions of the present disclosure can be
administered by any suitable route of administration. It will also
be appreciated that the preferred route will vary with the
condition and age of the recipient, and the disease being
treated.
[0164] For example, the compositions can be administered by oral,
parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV,
intracistemal injection or infusion, subcutaneous injection, or
implant), by inhalation spray nasal, vaginal, rectal, sublingual,
urethral (e.g., urethral suppository) or topical routes of
administration (e.g., gel, ointment, cream, aerosol, etc.) and can
be formulated, alone or together, in suitable dosage unit
formulations containing conventional non-toxic pharmaceutically
acceptable carriers, adjuvants, excipients, and vehicles
appropriate for each route of administration. Non-limiting examples
of carriers include phosphate buffered saline (PBS), saline or a
biocompatible matrix material such as a decellularized liver matrix
(DCM as disclosed in Wang et al. (2014) J. Biomed. Mater Res. A.
102(4):1017-1025) for topical or local administration. The
compositions can optionally contain a protease inhibitor, glycerol
and/or dimethyl sulfoxide (DMSO).
[0165] The compositions can be conveniently presented in dosage
unit form and can be prepared by any of the methods well known in
the art of pharmacy. The compositions can be, for example, prepared
by uniformly and intimately bringing the active ingredient into
association with a liquid carrier, a finely divided solid carrier
or both, and then, if necessary, shaping the product into the
desired formulation. In the composition the protein or peptide is
included in an amount sufficient to produce the desired therapeutic
effect. For example, pharmaceutical compositions of the disclosure
may take a form suitable for virtually any mode of administration,
including, for example, topical, ocular, oral, buccal, systemic,
nasal, injection, transdermal, rectal, and vaginal, or a form
suitable for administration by inhalation or insufflation.
[0166] Intranasal administration is a particularly preferred mode
of administration that includes administration via the nose, either
with or without concomitant inhalation during administration. Such
administration is typically through contact by the pharmaceutical
composition comprising the nanoemulsion composition with the nasal
mucosa, nasal turbinates or sinus cavity. Administration by
inhalation comprises intranasal administration, or may include oral
inhalation. Such administration may also include contact with the
oral mucosa, bronchial mucosa, and other epithelia.
[0167] However, the disclosure is not limited to intranasal
administration and pharmaceutical compositions of the disclosure
may be administered by alternative means, like oral or injectable
administration, as well. Useful injectable preparations include
sterile suspensions, solutions, or emulsions of the active
compound(s) in aqueous or oily vehicles. The compositions may also
contain formulating agents, such as suspending, stabilizing, and/or
dispersing agents. The formulations for injection can be presented
in unit dosage form, e.g., in ampules or in multidose containers,
and may contain added preservatives.
[0168] Compositions intended for oral use can be prepared according
to any method known to the art for the manufacture of
pharmaceutical compositions, and such compositions may contain one
or more agents selected from the group consisting of sweetening
agents, flavoring agents, coloring agents, and preserving agents in
order to provide pharmaceutically elegant and palatable
preparations. Tablets contain the active ingredient (including drug
and/or prodrug) in admixture with non-toxic pharmaceutically
acceptable excipients which are suitable for the manufacture of
tablets. These excipients can be for example, inert diluents, such
as calcium carbonate, sodium carbonate, lactose, calcium phosphate
or sodium phosphate; granulating and disintegrating agents (e.g.,
corn starch or alginic acid); binding agents (e.g., starch,
gelatin, or acacia); and lubricating agents (e.g., magnesium
stearate, stearic acid, or talc). The tablets can be left uncoated
or they can be coated by known techniques to delay disintegration
and absorption in the gastrointestinal tract and thereby provide a
sustained action over a longer period. For example, a time delay
material such as glyceryl monostearate or glyceryl distearate can
be employed. They may also be coated by the techniques described in
the U.S. Pat. Nos. 4,256,108; 4,166,452, and 4.265,874 to form
osmotic therapeutic tablets for control release. The pharmaceutical
compositions of the disclosure may also be in the form of
oil-in-water emulsions.
[0169] Liquid preparations for oral administration may take the
form of, for example, elixirs, solutions, syrups, or suspensions,
or they can be presented as a dry product for constitution with
water or other suitable vehicle before use. Such liquid
preparations can be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives, or hydrogenated
edible fats); emulsifying agents (e.g., lecithin, or acacia):
non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol,
Cremophore.TM., or fractionated vegetable oils); and preservatives
(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations may also contain buffer salts, preservatives,
flavoring, coloring, and sweetening agents as appropriate.
[0170] Exemplary dosage forms for pharmaceutical administration are
described herein. Examples include but are not limited to liquids,
ointments, creams, emulsions, lotions, gels, bioadhesive gels,
sprays, aerosols, pastes, foams, sunscreens, capsules,
microcapsules, suspensions, pessary, powder, semi-solid dosage
form, etc.
[0171] The immunogenic compositions can likewise be applied and/or
delivered utilizing electrophoretic delivery/electrophoresis.
Further, the compositions may be applied by a transdermal delivery
system such as a patch or administered by a pressurized or
pneumatic device (i.e., "gene gun"). Such methods, which comprise
applying an electrical current, are well known in the art.
[0172] The pharmaceutical compositions for administration may be
applied in a single administration or in multiple
administrations.
[0173] If applied topically, the compositions may be occluded or
semi-occluded. Occlusion or semi-occlusion may be performed by
overlaying a bandage, polyoleofin film, article of clothing,
impermeable barrier, or semi-impermeable barrier to the topical
preparation.
[0174] The present disclosure contemplates that many variations of
the describe compositions will be useful in the methods of the
present disclosure. To determine if a candidate composition is
suitable for pharmaceutical use, three criteria are analyzed. Using
the methods and standards described herein, candidate compositions
can be easily tested to determine if they are suitable. First, the
desired ingredients are prepared using the methods described
herein, to determine if a buffer-stabilized compositions can be
formed. If a buffer-stabilized compositions cannot be formed, the
candidate is rejected. Second, the candidate buffer-stabilized
compositions should be stable. A buffer-stabilized composition is
stable if it remains in solution, with the biological activity of a
protein or peptide preserved for a sufficient period to allow for
its intended use. For example, for pharmaceutical buffer-stabilized
compositions that are to be stored, shipped, etc., it may be
desired that the buffer-stabilized composition remain in solution
form for months to years. Typical buffer-stabilized compositions
that are relatively unstable, will lose their form within a day.
Third, the candidate pharmaceutical buffer-stabilized compositions
should have efficacy for its intended use. For example, the
pharmaceutical buffer-stabilized compositions disclosed herein
should induce a protective immune response or a therapeutic effect
to a detectable level.
[0175] The disclosed compositions can be provided in many different
types of containers and delivery systems. For example, in some
embodiments of the disclosed, the compositions are provided in a
cream or other solid or semi-solid form. The disclosed compositions
may be incorporated into hydrogel formulations.
[0176] The compositions can be delivered (e.g., to a subject or
customers) in any suitable container. Suitable containers can be
used that provide one or more single use or multi-use dosages of
the vaccines for the desired application. In some embodiments of
the disclosed, the compositions are provided in a suspension or
liquid form. Such compositions can be delivered in any suitable
container including spray bottles and any suitable pressurized
spray device. Such spray bottles may be suitable for delivering the
compositions intranasally or via inhalation. These containers can
further be packaged with instructions for use to form kits.
[0177] Formulation Techniques
[0178] Nanoemulsions of the present invention can be formed using
classic emulsion forming techniques. In brief, the oil phase is
mixed with the aqueous phase under relatively high shear forces
(e.g., using high hydraulic and mechanical forces) to obtain an
oil-in-water nanoemulsion. The emulsion is formed by blending the
oil phase with an aqueous phase on a volume-to-volume basis ranging
from about 1:9 to 5:1, preferably about 5:1 to 3:1, most preferably
4:1, oil phase to aqueous phase. The oil and aqueous phases can be
blended using any apparatus capable of producing shear forces
sufficient to form an emulsion such as French Presses or high shear
mixers (e.g., FDA approved high shear mixers are available, for
example, from Admix, Inc., Manchester, N.H.). Methods of producing
such emulsions are described in U.S. Pat. Nos. 5,103,497 and
4,895,452, and U.S. Patent Application Nos. 20070036831,
20060251684, and 20050208083, herein incorporated by reference in
their entireties.
[0179] In preferred embodiments, compositions used in the methods
of the present invention comprise droplets of an oily discontinuous
phase dispersed in an aqueous continuous phase, such as water. In
preferred embodiments, nanoemulsions of the present invention are
stable, and do not decompose even after long storage periods (e.g.,
greater than one or more years). Furthermore, in some embodiments,
nanoemulsions are stable (e.g., in some embodiments for greater
than 3 months, in some embodiments for greater than 6 months, in
some embodiments for greater than 12 months, in some embodiments
for greater than 18 months) after combination with an immunogen. In
preferred embodiments, nanoemulsions of the present invention are
non-toxic and safe when administered (e.g., via spraying or
contacting mucosal surfaces, swallowed, inhaled, etc.) to a
subject.
[0180] In some embodiments, a portion of the emulsion may be in the
form of lipid structures including, but not limited to,
unilamellar, multilamellar, and paucliamellar lipid vesicles,
micelles, and lamellar phases.
[0181] In general, the preferred non-toxic nanoemulsions are
characterized by the following: they are approximately 200-800 nm
in diameter, although both larger and smaller diameter
nanoemulsions are contemplated; the charge depends on the
ingredients; they are stable for relatively long periods of time
(e.g., up to two years); they are non-irritant and non-toxic
compared to their individual components due, at least in part, to
their oil contents that markedly reduce the toxicity of the
detergents and the solvents; they are effective at concentrations
as low as, for example, 0.1%.
[0182] The present invention is not limited by the type of subject
administered (e.g., in order to stimulate an immune response (e.g.,
in order to generate protective immunity (e.g., mucosal and/or
systemic immunity))) a composition of the present invention.
Indeed, a wide variety of subjects are contemplated to be benefited
from administration of a composition of the present invention. In
preferred embodiments, the subject is a human. In some embodiments,
human subjects are of any age (e.g., adults, children, infants,
etc.) that have been or are likely to become exposed to a
microorganism (e.g., a pathogenic microorganism). In some
embodiments, the human subjects are subjects that are more likely
to receive a direct exposure to pathogenic microorganisms or that
are more likely to display signs and symptoms of disease after
exposure to a pathogen (e.g., immune suppressed subjects). In some
embodiments, the general public is administered (e.g., vaccinated
with) a composition of the present invention (e.g., to prevent the
occurrence or spread of disease). For example, in some embodiments,
compositions and methods of the present invention are utilized to
vaccinate a group of people (e.g., a population of a region, city,
state and/or country) for their own health (e.g., to prevent or
treat disease). In some embodiments, the subjects are non-human
mammals (e.g., pigs, cattle, goats, horses, sheep, or other
livestock; or mice, rats, rabbits or other animal). In some
embodiments, compositions and methods of the present invention are
utilized in research settings (e.g., with research animals).
[0183] In preferred embodiments, an immunogenic composition of the
present invention comprises a suitable amount of the
antigen/immunogen to induce an immune response in a subject when
administered to the subject. In preferred embodiments, the immune
response is sufficient to provide the subject protection (e.g.,
immune protection) against a subsequent exposure to the immunogen
or the microorganism (e.g., bacteria or virus) from which the
immunogen was derived. The present invention is not limited by the
amount of immunogen used. In some preferred embodiments, the amount
of immunogen (e.g., protein antigen) in an immunogenic composition
(e.g., for use as an immunization dose) is selected as that amount
which induces an immunoprotective response without significant,
adverse side effects. The amount will vary depending upon which
specific immunogen or combination thereof is/are employed, and can
vary from subject to subject, depending on a number of factors
including, but not limited to, the species, age and general
condition (e.g., health) of the subject, and the mode of
administration. Procedures for determining the appropriate amount
of immunogen administered to a subject to elicit an immune response
(e.g., a protective immune response (e.g., protective immunity)) in
a subject are well known to those skilled in the art.
[0184] In some embodiments, it is expected that each dose (e.g., of
an immunogenic composition (e.g., administered to a subject to
induce an immune response (e.g., a protective immune response
(e.g., protective immunity))) comprises 0.05-5000 .mu.g of each
immunogen (e.g., recombinant and/or purified protein), in some
embodiments, each dose will comprise 1-500 .mu.g, in some
embodiments, each dose will comprise 350-750 .mu.g, in some
embodiments, each dose will comprise 50-200 .mu.g, in some
embodiments, each dose will comprise 25-75 .mu.g of immunogen
(e.g., recombinant and/or purified protein). In some embodiments,
each dose comprises an amount of the immunogen sufficient to
generate an immune response. An effective amount of the immunogen
in a dose need not be quantified, as long as the amount of
immunogen generates an immune response in a subject when
administered to the subject. An optimal amount for a particular
administration (e.g., to induce an immune response (e.g., a
protective immune response (e.g., protective immunity))) can be
ascertained by one of skill in the art using standard studies
involving observation of antibody titers and other responses in
subjects.
[0185] In some embodiments, it is expected that each dose (e.g., of
an immunogenic composition (e.g., administered to a subject to
induce and immune response)) is from 0.001 to 15% or more (e.g.,
0.001-10%, 0.5-5%, 1-3%, 2%. 6%, 10%, 15% or more) by weight
immunogen (e.g., neutralized bacteria or virus, or recombinant
and/or purified protein). In some embodiments, an initial or prime
administration dose contains more immunogen than a subsequent boost
dose.
[0186] In some embodiments, an immunogenic composition of the
present invention is formulated in a concentrated dose that can be
diluted prior to administration to a subject. For example,
dilutions of a concentrated composition may be administered to a
subject such that the subject receives any one or more of the
specific dosages provided herein. In some embodiments, dilution of
a concentrated composition may be made such that a subject is
administered (e.g., in a single dose) a composition comprising
about 0.1-50% of the nanoemulsion adjuvant present in the
concentrated composition. In some preferred embodiments, a subject
is administered in a single dose a composition comprising 1% of the
NE present in the concentrated composition. Concentrated
compositions are contemplated to be useful in a setting in which
large numbers of subjects may be administered a composition of the
present invention (e.g., an immunization clinic, hospital, school,
etc.). In some embodiments, an immunogenic composition of the
present invention (e.g., a concentrated composition) is stable at
room temperature for more than 1 week, in some embodiments for more
than 2 weeks, in some embodiments for more than 3 weeks, in some
embodiments for more than 4 weeks, in some embodiments for more
than 5 weeks, and in some embodiments for more than 6 weeks.
[0187] Generally, an immunogenic composition comprising an emulsion
of the invention will comprise at least 0.0010% to 100%, preferably
0.01 to 90%, of emulsion per ml of liquid composition. It is
envisioned that the formulations may comprise about 0.001%, about
0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.025%,
about 0.05%, about 0.075%, about 0.1%, about 0.25%, about 0.5%,
about 1.0%, about 2.5%, about 5%, about 7.5%, about 10%, about
12.5%, about 15%, about 20%, about 25%, about 30%, about 35%, about
40%, about 50%, about 55%, about 60%, about 65%, about 70%, about
75%, about 80%, about 85%, about 90%, about 95% or about 100% of
emulsion per ml of liquid composition. It should be understood that
a range between any two figures listed above is specifically
contemplated to be encompassed within the metes and bounds of the
present invention. Some variation in dosage will necessarily occur
depending on the condition of the specific pathogen and the subject
being immunized.
[0188] In some embodiments, following an initial administration of
a composition of the present invention (e.g., an initial
vaccination), a subject may receive one or more boost
administrations (e.g., around 2 weeks, around 3 weeks, around 4
weeks, around 5 weeks, around 6 weeks, around 7 weeks, around 8
weeks, around 10 weeks, around 3 months, around 4 months, around 6
months, around 9 months, around 1 year, around 2 years, around 3
years, around 5 years, around 10 years) subsequent to a first,
second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth,
and/or more than tenth administration. Although an understanding of
the mechanism is not necessary to practice the present invention
and the present invention is not limited to any particular
mechanism of action, in some embodiments, reintroduction of an
immunogen in a boost dose enables vigorous systemic immunity in a
subject. The boost can be with the same formulation given for the
primary immune response, or can be with a different formulation
that contains the immunogen. The dosage regimen will also, at least
in part, be determined by the need of the subject and be dependent
on the judgment of a practitioner.
[0189] Dosage units may be proportionately increased or decreased
based on several factors including, but not limited to, the weight,
age, and health status of the subject. In addition, dosage units
may be increased or decreased for subsequent administrations (e.g.,
boost administrations).
[0190] It is contemplated that the compositions and methods of the
present invention will find use in various settings, including
research settings. For example, compositions and methods of the
present invention also find use in studies of the immune system
(e.g., characterization of adaptive immune responses (e.g.,
protective immune responses (e.g., mucosal or systemic immunity))).
Uses of the compositions and methods provided by the present
invention encompass human and non-human subjects and samples from
those subjects, and also encompass research applications using
these subjects. Compositions and methods of the present invention
are also useful in studying and optimizing nanoemulsions,
immunogens, and other components and for screening for new
components. Thus, it is not intended that the present invention be
limited to any particular subject and/or application setting.
[0191] In some embodiments, the present invention provides a kit
comprising an immunogenic composition. In some embodiments, the kit
further provides a device for administering the composition. The
present invention is not limited by the type of device included in
the kit. In some embodiments, the device is configured for nasal
application of the composition of the present invention (e.g., a
nasal applicator (e.g., a syringe) or nasal inhaler or nasal
mister). In some embodiments, a kit comprises a composition
comprising a nanoemulsion adjuvant in a concentrated form (e.g.,
that can be diluted prior to administration to a subject).
[0192] In some embodiments, all kit components are present within a
single container (e.g., vial or tube). In some embodiments, each
kit component is located in a single container (e.g., vial or
tube). In some embodiments, one or more kit component are located
in a single container (e.g., vial or tube) with other components of
the same kit being located in a separate container (e.g., vial or
tube). In some embodiments, a kit comprises a buffer. In some
embodiments, the kit further comprises instructions for use.
EXAMPLES
[0193] The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof.
[0194] In the experimental disclosure which follows, the following
abbreviations apply: eq (equivalents); .mu. (micron); M (Molar);
.mu.M (micromolar): mM (millimolar); N (Normal); mol (moles); mmol
(millimoles); .mu.mol (micromoles); nmol (nanomoles); g (grams); mg
(milligrams); .mu.g (micrograms): ng (nanograms); L (liters); ml
(milliliters); .mu.l (microliters); cm (centimeters); mm
(millimeters): .mu.m (micrometers); nM (nanomolar):.degree. C.
(degrees Centigrade); and PBS (phosphate buffered saline).
Example 1
rH5 Antigen, QS-21, CpG and Nanoemulsion
[0195] A series of experiments were generated and conducted in
order to identify and characterize the adjuvant properties of
various different emulsion delivery systems in combination with
different immunostimulatory compounds. In particular, experiments
were conducted during development of embodiments of the invention
in order to evaluate the combination of 3 different adjuvants, a
TLR9 agonist, composed of synthetic oligodeoxynucleotides (ODN)
containing immunostimulatory CpG motifs (CpG), a QS-21 and
nanoemulsion adjuvant, composed of two surfactants, oil, and
organic solvent, which possesses both immunostimulatory and
delivery properties. While individual adjuvant members have
previously been documented to be effective adjuvants, there exists
no report or data in the field indicating what might happen to the
adjuvant properties of each individual adjuvant member if the
members are combined.
[0196] Accordingly, combinations of various TLR agonists and
nanoemulsion adjuvant (NE) formulations were evaluated for adjuvant
activity when used in combination with antigen/immunogen as
described below.
[0197] Materials and Methods. rH5 was obtained from Fraunhofer USA
Center for Molecular Biotechnology. Newark Del. Briefly, the
recombinant rH5 protein was expressed in tobacco plants by
agrobacterium gene transfer. The transgene encoding amino acids
17-532 of the HA gene of the A/Indonesia/05/2005 (H5N1) virus was
cloned into a plasmid vector. This vector was transformed into
Agrobacterium tumefaciens. The transformed bacteria were cultured
overnight and subsequently vacuum infiltrated into 6-week-old
Nicotiana benthamiana plants. After seven days, leaves were
harvested and homogenized, the extracts were clarified by
centrifugation, and the HA protein was purified by immobilized
metal affinity chromatography and anion exchange
chromatography.
[0198] Purified QS-21 was purchased from Desert King International
(San Diego, Calif., USA). ODN 1826 is a CpG oligodeoxynucleotides
(ODN) class B ODN specific for murine TLR9. B-class CpG ODNs
contain a full phosphorothioate backbone with one or more CpG
dinucleotides. They strongly activate B cells but weakly stimulate
IFN-.alpha. secretion. ODN 1826 VACCIGRADE.TM. is a preclinical
grade preparation of the CpG ODN 1826. It is tested for sterility
and the presence of endotoxins. ODN 1826 VACCIGRADE.TM. is
guaranteed sterile and its endotoxin level is <1 EU/mg
(measurement by kinetic chromogenic LAL assay). ODN1826 sequence:
5'-tccatgacgttcctgacgtt-3' (20 mer) was purchased from In vivo Gen
(San Diego, Calif., USA).
[0199] Phosphate-buffered saline was purchased from Sigma Chemical
Company (Saint Louis, Mo., USA).
[0200] The nanoemulsion adjuvants were formed by emulsification of
an oil, purified water, nonionic detergent, organic solvent and
surfactant, such as a cationic surfactant. An exemplary specific
nanoemulsion adjuvant is designated as "60% W805EC" or 60% DODAC
NE". The 60% W805EC-vaccine adjuvant or 60% DODAC-vaccine adjuvant
is composed of the ingredients shown in Table 3 below: purified
water, USP; soybean oil USP; Dehydrated Alcohol, USP [anhydrous
ethanol]; Polysorbate 80, NF, cetylpyridinium chloride. USP (CPC)
and/or dioctadecyldimethylammonium chloride (DODAC). All components
of this exemplary nanoemulsion adjuvant are included on the FDA
list of approved inactive ingredients for Approved Drug
Products.
TABLE-US-00003 TABLE 3 Composition of 60% W805EC-Adjuvant (w/w %)
60% 60% 60% DODAC DODAC/ Ingredients W.sub.80SEC NE CPCNE Purified
Water, USP 54.10% 54.10% 54.10% Soybean Oil, USP 37.67% 37.67%
37.67% Dehydrated Alcohol, USP 4.04% 4.04% 4.04% (anhydrous
ethanol) Polysorbate 80, NF 3.55% 3.55% 3.55% Cetylpyridinium
Chloride 0.64% -- 0.32% (CPC), USP Dioctadecyldimethylammonium --
0.64% 0.32% chloride (DODAC)
[0201] Combinations of various TLR agonists and nanoemulsion
adjuvant (NE) formulations were generated and tested for adjuvant
activity. Specifically, formulations were prepared and tested for
potential adjuvant activity for inducing immune responses upon an
intramuscular route of administration. Immune responses
analyzed/characterized included production of
hemagglutination-inhibition (HAI) antibody titers and viral
neutralizing (VN) antibody titers, both of which provide an
indication of the functional components of an induced immune
response. Various formulations were generated (See Table 4) and
tested. It is noted that a cut-off for geometric mean titer (GMT)
of .gtoreq.1:40 for HAI is accepted in the art as a correlate of
protection against influenza.
TABLE-US-00004 TABLE 4 Evaluation of W805EC adjuvant in combination
with QS21 or CpG ODN, or using nanoemulsions formulated with DODAC
cationic surfactant by the intramuscular route in CD-1 mice. Amount
Additional (.mu.g) Adjuvant of rH5 amount antigen (.mu.g) per per
50 .mu.L 50 .mu.L IM Group/F# Description IM dose dose First Set
Exploratory Formulations 1 QS-21 (alone) 10 .mu.g QS-21: 10 .mu.g 2
5% W.sub.805EC + QS-21 10 .mu.g QS-21: 10 .mu.g 3 5% DODAC NE 10
.mu.g -- 4 5% DODAC/CPC NE 10 .mu.g -- 5 CpG (alone) 10 .mu.g CpG:
10 .mu.g 6 5% W.sub.805EC + CpG 10 .mu.g CpG: 10 .mu.g 7 rH5 10
.mu.g 0 8 No Treatment (Naive) 0 0 Second Set of Exploratory
Formulations 1 5% W.sub.805EC + CpG 10 .mu.g CpG: 20 .mu.g 2 5%
DODAC NE + CpG 10 .mu.g CpG: 20 .mu.g 3 5% DODAC NE 10 .mu.g -- 4
CpG (alone) 10 .mu.g CpG: 20 .mu.g 5 5% DODAC NE + CpG 10 .mu.g
CpG: 10 .mu.g 6 CpG (alone) 10 .mu.g CpG: 10 .mu.g 7 5% DODAC NE +
CpG 10 .mu.g CpG: 5 .mu.g 8 CpG (alone) 10 .mu.g CpG: 5 .mu.g 9 5%
DODAC NE + CpG 5 .mu.g CpG: 10 .mu.g 10 CpG (alone) 5 .mu.g CpG: 10
.mu.g
[0202] Animals were vaccinated as follows: PBS; rH5: (10
.mu.g/animal). B animals per group/64 animals, Three IM
immunizations each delivered in a volume of 50 01 at 0, 2, and 4
wks; Sacrifice at week 6. Briefly, the mixing protocol for a 700
.mu.L batch size of a test vaccine formulation comprised of mixing
three or four components, the volume of each constituent in each
test formulations is shown in Table 5. A PBS solution was mixed
gently with 5.8 mg/mL rH5 antigen solution. The nanoemulsion
adjuvant, either 60% W85EC, 60 DODAC/CPC NE or 60% DODAC NE was
than added and mixed gently. CpG (1 mg/mi) solution or QS-21 (1
mg/ml) solution was added last and mixed gently. The test vaccine
formulation contained rH5 antigen and 5% nanoemulsion adjuvant with
or without CpG or QS-21 in PBS (7.times.).
TABLE-US-00005 TABLE 5 Composition of vaccine formulations of
nanoemulsion adjuvant in combination with QS-21 or CpG ODN. Volume
of Volume 1 mg/ml of 5.8 CpG or mg/mL Volume 1 mg/ml Volume rH5 of
QS-21 of PBS solution 60% NE solution (.mu.l) in (.mu.L) in (.mu.L)
in (.mu.L) in Group Description 700 .mu.L 700 .mu.L 700 .mu.L 700
.mu.L First Set Exploratory Formulations 1 QS-21 (alone) 536 24 --
140 2 5% W.sub.805EC + QS-21 478 24 58 140 3 5% DODAC NE 618 24 58
-- 4 5% DODAC/CPC NE 618 24 58 -- 5 CpG (alone) 396 24 0 280 6 5%
W.sub.805EC + CpG 338 24 58 280 7 rH5 676 24 -- -- 8 No Treatment
(Naive) -- -- -- -- Second Set of Exploratory Formulations 1 5%
W.sub.805EC + CpG 338 24 58 280 2 5% DODAC NE + CpG 338 24 58 280 3
5% DODAC NE 618 24 58 -- 4 CpG (alone) 396 24 58 280 5 5% DODAC NE
+ CpG 478 24 58 140 6 CpG (alone) 536 24 58 140 7 5% DODAC NE + CpG
548 24 58 70 8 CpG (alone) 606 24 -- 70 9 5% DODAC NE + CpG 490 12
58 140 10 CpG (alone) 548 12 -- 140
[0203] Vaccinations. Six- to eight-week-old female CD-1 mice (6-8
wk; n=6-10/group) (Charles River Laboratories) were immunized twice
(FIG. 9) or three times (FIG. 1) intramuscularly at 2 week
intervals with 50 .mu.L of test vaccine formulations. All test
vaccine formulations at a dosing volume of 50 .mu.L were
administered by intramuscular (IM) injection in the left tibialis
anterior (TA) muscle of mice lightly anaesthetized with Isoflurane
(CDMV, Saint Hyacinthe, QC).
[0204] Mice were immunized at the intervals described above using
rH5 antigen, either alone, or co-formulated with CpG, nanoemulsion,
or nanoemulsion+CpG as described in Table 4. Naive animals were
used for comparison as controls.
[0205] Immune response assays. Animals were bled from the saphenous
vein at various timepoints after immunization and antigen-specific
total IgG, was measured in serum by endpoint ELISA (in triplicate)
for individual animals, using 96-well plates coated with rHA (1
.mu.g/well).
[0206] rH5-specific IgG ELISA. Serum was obtained from the
saphenous vein every 2-weeks post-initial immunization, and
anti-rH5 specific IgG end-titers were measured by ELISA. Briefly,
serum samples were serially diluted in PBS with 0.1% BSA, and
incubated on microtiter plates coated with 1 .mu.g/mL rH5. ELISAs
were developed with an alkaline phosphatase detection system, and
quantified by measuring the optical density (OD) at 405 nm (OD405).
Endpoint titers are reported as the reciprocal of the highest serum
dilution giving an OD above a cutoff value (sum of OD of the same
dilution of a control serum from an untreated mouse and two times
the standard deviation).
[0207] Hemagglutination inhibition assay. Mouse serum samples from
week 6 were analyzed using the HAI assay. Prior to analysis, serum
samples were treated with receptor-destroying enzyme (RDE,
Denka-Seiken) at 37.degree. C. followed by heat inactivation of RDE
at 56.degree. C. and storage at 4.degree. C. during analysis. The
assays used A/Indonesia/05/05 (xPR8 IBCDC RG-2 from CDC) virus and
1% horse red blood cells. Serum dilutions started at 1:20 and
samples without detectable HAI activity were assigned a value of
10.
[0208] Cell-mediated immunity. Spleens were removed aseptically at
the time of sacrifice 2 weeks following the final immunization.
Splenocytes were pelleted by centrifugation at 2000 rpm for 5 min
and resuspended in ACK lysis buffer for <3 minutes to remove red
blood cells (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). PBS was added
to stop lysis, and cells were pelleted and washed again in PBS. The
cell pellet was then resuspended in T-Cell media (DMEM supplemented
with 5% FBS, 2 mM L-glutamine, 1.times. nonessential amino acids, 1
mM sodium pyruvate, 10 mM MOPS, 50 .mu.M 2-mercaptoethanol, 100 IU
penicillin, and 100 .mu.g/mL streptomycin and filtered through a
cell strainer. Splenocytes were then plated at a density of
4.times.10.sup.5 cells/well in 96-well tissue culture plates and
stimulated using 5 .mu.g/ml rH5 antigen or medium alone. Cell-free
supernatants were harvested after 72 hours culture at 37.degree. C.
Supernatants were stored at -80.degree. C. prior to Luminex
multiplex analysis (Millipore) to determine cytokine profiles using
selected kits according to manufacturers' instructions (Milliplex
Catalog ID.MCYTOMAG-70K-08.Mou).
[0209] Statistical Analysis. Data were analyzed using Graph-Pad
Prism (GraphPad Software, San Diego. Calif.). Statistical
significance of the difference between the two groups was
calculated by Student's 2-tailed I-test and between three or more
groups by I-factor analysis of variance (ANOVA) followed by post
hoc analysis.
[0210] Analytical Methods Used in the Short Term Stability Studies.
The various formulations were filled into 1.8 mL Type 1 glass vials
with a PTFE-lined screw cap. The stability parameters assessed for
these formulations were physical appearance, pH, mean particle
size, SDS-Page Gel for 70-80 kd. Dynamic light scattering using the
Malvern Zetasizer was used to determine particle size, particle
size distribution profiles and a polydispersity index. Acceptable
criteria developed and considered for each criteria are depicted in
Table 6.
TABLE-US-00006 TABLE 6 Test method and acceptance criteria for the
formulations placed on informal stability. Acceptance Criteria for
Each Formulation Type Stability Control Solutions 20% W.sub.805EC +
Parameter Test Method rH5 + CpG rH5 +/- CpG Physical Appearance
Visual No Precipitation and/or Cloudy Solution No Phase Separation
pH pH Meter +/-0.5 +/-0.5 Particle Dynamic -- Z-Ave: +/-200 nm Size
Light Scattering PdI Dynamic Light Scattering Less than 0.25 Less
than 0.25 rH5 SDS Page Band Band Integrity Gel Present Present
(kDa) Profile Profile compares to compares to reference reference
material material rH5 SRID Concentration Concentration Potency
compares to compares to reference reference material material
[0211] Physical Appearance Test. Observations of physical
appearance were recorded according to the nanoemulsion stability
assessment criteria shown in Table 7. Physical appearance of the
formulations was determined at the initial time point and upon
various storage conditions.
TABLE-US-00007 TABLE 7 Nanoemulsion Stability Parameters,
Description and Acceptance Criteria pH Assessment: The pH was
measured using a standard pH meter with the appropriate probe that
was used. Stability Parameter Description Acceptance Criteria Color
A white to off white liquid A white to off white liquid acceptable.
Yellow (light to dark), tan, and shades of brown not acceptable.
Creaming A white, creamy layer on top of the emulsion that is more
opaque than the rest of the emulsion. Remixing will restore
homogeneity. Presence (+) or absence (-) All stages of creaming are
acceptable Settling A gradual decrease in opacity of the emulsion
from top to bottom. Remixing will restore homogeneity.
Mild-cloudiness gradient from top to bottom (no defining layers)
Moderate-clear at bottom of vial with increasing opacity toward the
cap Severe-3-4 distinct layers Extreme-only 2 layers All stages of
settling are acceptable Phase Separation Separation of the oil and
water phases of the emulsion. Remixing will not restore
homogeneity. Pass: None Insignificant- a few droplets are visible
at surface Mild-oil layer equals <1% of total height Fail:
Moderate-a film of oil >1% of emulsion height Severe-3 distinct
layers Extreme-total separation into 2 phases (oil and water)
[0212] Mean Particle Size Analysis and Polydispersity Index (PdI):
The mean particle size (Z-average) and polydispersity index (PdI)
were determined for all the stability samples. The particle size
and PdI of the sample was measured by photon correlation
spectroscopy using a Malvern Zetasizer Nano ZS90 (Malvern
Instruments, Worcestershire, UK), according to the Malvern user's
manual for Particle Sizing (Malvern). All measurements were carried
out at 25.degree. C. after dilution to 1% nanoemulsion with sterile
water. The aqueous systems were not diluted.
[0213] Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
Method for rH5: The nanoemulsion vaccine samples containing rH5
antigen, or rH5 antigen alone, were prepared by acetone
precipitation at (-20.degree.) C for 60 minutes to remove
nanoemulsion while precipitating the antigen for subsequent
SDS-PAGE analysis (Invitrogen Xcell mini-gel electrophoresis
System). After precipitation, the samples were spun for 15 minutes
at 14,000.times.g and the supernatant was removed. The slightly
dried pellet was resuspended in 50 ul of 1.times. sample buffer
(Thermo 39000, lot# PJ207541) and incubated at 4.degree. C. for 2
hours. The samples next were mixed by pipeting up and down followed
by -20.degree. C. storage. Samples (5 ul per lane, 1 ug rH5 antigen
per lane) were loaded onto a Criterion.TM. XT, 12% Bis-Tris, 18
well, gel (BioRad Cat#345-0118) and run at 120 volts using XT MES,
(Biorad 161-0789) running buffer unit the dye front reached the end
of the gel. Gels were stained at room temperature for 2 hours using
Imperial.TM. Protein Stain, (ThermoScientific, Cat#24615), followed
by destaining overnight in water. Precision Plus markers (BioRad)
were used as a standard to assess molecular weight.
[0214] Formulations as shown in Table 8 were administered to mice
according to the schedule shown in FIG. 1.
TABLE-US-00008 TABLE 8 Evaluation of W.sub.805EC adjuvant in
combination with QS21 or CpG ODN, or using nanoemulsions formulated
with DODAC cationic surfactant by the intramuscular route in CD-1
mice. Amount Adjuvant of rH5 System per each Concentration
Immunization or Amount (.mu.g per 50 (.mu.g) per Group Description
.mu.L dose) 50 .mu.L IM dose 1 QS-21 10 QS-21 (10 .mu.g) 2 NE +
QS-21 10 5% W.sub.805EC NE + QS-21 (10 .mu.g) 3 DODAC NE 10 5%
DODAC NE 4 DODAC/CPC NE 10 5% DODAC/CPC NE 5 CpG 10 CpG (10 .mu.g)
6 NE + CpG 10 5% W.sub.805EC NE + CpG (20 .mu.g) 7 Antigen Alone 10
-- 8 Naive 0 --
[0215] rH5-specific serum IgG in treatment groups assessing a
single cationic DODAC formulation (Group 3) were compared to dual
cationic formulation (5% DODAC/CPC NE) (Group 4) using serum
obtained 2 weeks following the second intramuscular vaccination
(See FIG. 2). Interestingly, the geometric mean titer (GMT) of
rH5-specific serum IgG was highest in the group immunized using the
DODAC single-cationic formulation (GMT: 1.17.times.10.sup.6)
compared to W.sub.805EC (GMT: 1.65.times.10.sup.5) (P=0.015). The
dual cationic combination of CPC+DODAC (GMT. 7.61.times.10.sup.5)
was not significantly different than DODAC alone (GMT:
1.17.times.10.sup.6). Addition of the CPC cationic surfactant to
the DODAC formulation did not enhance the cytokine response
compared to DODAC alone (See FIG. 3).
[0216] Groups of CD-1 mice were immunized intramuscularly in a
series of three vaccinations at 2 week intervals. Mice were
sacrificed 2 weeks after the final immunization (Week 6) for
evaluation of serum antibodies and rH5-specific cytokine release by
spleen cells.
[0217] The DODAC formulation (DD) stimulated a more rapid
rH5-specific IgG antibody response (Week 4) (See FIG. 4-A) and
higher "functional" HAI antibody responses at Week 6 (See FIG. 4-B)
compared to W805EC. The DODAC formulation stimulated a geometric
mean HAI titer of 1:111 (7/8 animals.gtoreq.1:40) compared to only
1:32 for W805EC (4/8 animals.gtoreq.1:40). A cut-off for geometric
mean titer (GMT) of .gtoreq.1:40 for HAI is accepted as a correlate
of protection for influenza.
[0218] An adjuvant formulation comprising W805EC nanoemulsion
together with CpG ODN 1826, a TLR9 agonist, was also evaluated.
CD-1 mice were immunized IM at 2 week intervals using nanoemulsion
plus 10 .mu.g rH5 antigen. As shown in FIG. 5, 5% W805EC in
combination with 20 .mu.g CpG ODN and 10 .mu.g rH5 antigen
stimulated high levels (GMT>107) of rH5-specific IgG in serum
after 3 immunizations (Week 6)(See FIG. 5A). The combination of
W805EC+CpG also activated the highest levels of "functional" HAI
antibody (GMT: 160; 75% responders) when compared to W805EC alone
(GMT: 32; 50% responders), or CpG alone (GMT: 77; 50%
responders)(See FIG. 5B).
[0219] The profile of cell-mediated immunity (Th1, Th2, and Th17)
was determined by cytokines secretion analysis upon rH5 antigen
re-stimulation of spleen cells obtained at the time of sacrifice on
week 6 from mice immunized intramuscularly using the new
nanoemulsion-formulations (See FIG. 6). Surprisingly, W805EC
nanoemulsion in combination with CpG synergistically increased Th1
and Th17 responses while at the same time reducing or "shifting"
the cellular response away from Th2 responses with concurrent
retention of IL-10 to provide anti-inflammatory activity (See FIG.
6).
[0220] Overall, the combination of W.sub.805EC+CpG activated a
distinct profile characterized by strikingly greater production of
Th1 cytokines (IFN.gamma., IL-2 and TNF.alpha.) when compared to
either W805EC or CpG alone (FIG. 6 A-C).
[0221] In contrast to CpG ODN, addition of QS21 to the W805EC
nanoemulsion formulation did not enhance serum rH5-specific IgG
(See FIG. 7A), HAI antibody responses (SEE FIG. 7B) or
cell-mediated immune responses (cytokine profile) (See FIG. 7) when
compared to QS21 alone at a dose of 10 .mu.g. QS21 stimulated high
levels of HAI antibody (GMT 1:386) together with a strong and
balanced Th1 plus Th2 cytokine profile (See FIG. 8).
[0222] Additional adjuvant formulations were formulated (See Table
9, below), administered via an intramuscular route of
administration and analyzed for production of "functional" HAI and
VN antibodies. Formulations are shown in Table 9 below using a
combination of 5% W.sub.805EC or 5% DODAC NE adjuvant together with
immune modulators CpG at varying doses as shown in Table 9 and FIG.
8.
TABLE-US-00009 TABLE 9 Evaluation of W.sub.805EC adjuvant in
combination with QS21 or CpG ODN, or using nanoemulsions formulated
with DODAC cationic surfactant by the intramuscular route in CD-1
mice. Adjuvant Amount System of rH5 Concentration per each or
Amount Immunization (.mu.g) per (.mu.g per 50 .mu.L Group
Description 50 .mu.L dose) IM dose 1 5% W.sub.805EC 10 CpG (20
.mu.g) 2 5% DODAC NE + CpG 10 CpG (20 .mu.g) 3 5% DODAC NE 10 -- 4
CpG 10 CpG (20 .mu.g) 5 5% DODAC NE + CpG 10 CpG (10 .mu.g) 6 CpG
10 CpG (10 .mu.g) 7 5% DODAC NE + CpG 10 CpG (5 .mu.g) 8 CpG 10 CpG
(5 .mu.g) 9 5% DODAC NE + CpG 5 CpG (10 .mu.g) 10 CpG 5 CpG (10
.mu.g)
[0223] Single-radial immunodiffusion (SRID) analysis. This
procedure was used to assess the antigen stability of nanoemulsion
adjuvanted rH5 vaccines. The haemagglutinin (HA) concentration of
stored vaccines was evaluated by SRID analysis with comparison to
an rH5 antigen control in which the HA concentration is known.
Briefly, 1% agarose is prepared using 1.times.PBS, pH 7.2. The
anti-HA antibodies are added to the agarose and gently transferred
to a gel bond using a sterile pipette. The analysis utilized sheep
anti-rH5 (lot CRP 13-02-G765) developed at Fraunhofer USA Center
for Molecular Biotechnology (FhCMB). The reference and test
vaccines are diluted in PBS to achieve a range of rH5
concentrations as shown in FIG. 13. After solidification, 4 mm
wells are prepared using a gel punch and 20 .mu.l of the
appropriate vaccine dilution is added to each well. The plates are
incubated in a scaled humidity chamber at room temperature for
18-24 hours. After incubation, plates are submerged in sterile
saline (20 minutes), rinsed for 10 minutes in purified water, dryed
on filter paper (2-6 hours), stained for 7-10 minutes, and
destained for 4-5 minutes. Gel images are scanned and stored as a
digital image for subsequent measurement of ring diameter in two
dimensions, rH5 concentration is calculated based on orthogonal
measurements of the diameters for each duplicate well and the
dilution factors for both the reference and the samples using an
Excel SRID Trivalent Calculation Spreadsheet as determined by the
parallel line assay method. Stability Results of Appearance, pH,
Particle Size Analysis, Polydispersity Index (PdI) of NE adjuvant
stability and formulation of combination vaccines. The stability of
NE+CpG ODN vaccines (See Table 10, below) was assessed based on
physical-chemical parameters after storage for 3 days or 14 days at
5.degree. C. (See Tables 10-11, and FIGS. 10-12). In addition, as
shown in FIG. 13, evaluation of rH5 antigen in vaccine formulations
designated 1-4, (Table 9) by single-radial immuno-diffusion
analysis (SRID) demonstrated that there was no loss of antigen
immunoreactivity or concentration (rH5 concentration of 200
.mu.g/ml) after storage of the vaccines for 3-14 days at 5.degree.
C.
[0224] Thus, the invention provides adjuvant formulations that
display excellent short-term nanoemulsion stability and
compatibility with CpG ODN as required for extemporaneous
preparation of the vaccines.
TABLE-US-00010 TABLE 10 Control formulations compared to the test
formulations with rH5 and CpG in a buffered solution. Storage Lot #
Lot # Condition Appearance pH Nanoemulsion Adjuvant 60%
DODAC/Tween80 X-17768 5.degree. C. Pass 5.045 Antigen Control rH5
Eng. Lot -70.degree. C. Pass 7.314 Adjuvant Control CpG Adjuvant
-70.degree. C. Pass 6.274 F 4 CpG (20 .mu.g) + rH5 (10 .mu.g)
X-1859 X-1845 Initial 14 days @ 5.degree. C. Pass Pass 7.435 7.576
F 6 CpG (10 .mu.g) + rH5 (10 .mu.g) X-1861 X-1847 Initial 14 days @
5.degree. C. Pass Pass 7.528 7.714 F 8 CpG (5 .mu.g) + rH5 (10
.mu.g) X-1863 X-1849 Initial 14 days @ 5.degree. C. Pass Pass 7.512
7.697 F 10 CpG (10 .mu.g) + rH5 (5 .mu.g) X-1865 X-1851 Initial 14
days @ 5.degree. C. Pass Pass 7.531 7.705
TABLE-US-00011 TABLE 11 Evaluation of appearance, pH, particle size
and zeta potential of NE + CpG ODN combination vaccines after
storage at 5.degree. C. Storage Particle Lot # Lot # Condition
Appearance pH Size (nm) Pdl DPI 0% DODAC in X-1776B (5.degree. C.)
Pass 5.045 414.7 .+-. 2.1 0.160 .+-. 0.021 Water Control Control:
5% X-18 8 Initial Pass 7.444 40 .0 .+-. 4.1 0.178 .+-. 0.017 DODAC
in PBS X-18 8 3 days @ 5.degree. C. Pass 7.515 3 1.4 .+-. .3 0.175
.+-. 0.005 (no rH5, No CpG) TBD 14 days @ 5.degree. C. F 1 5%
W.sub.80 EC + CpG X-1856 Initial Pass 7.462 369. .+-. 2.3 0.18 .+-.
0.007 (20 .mu.g) + rH5 (10 .mu.g) X-1 42 14 days @ 5.degree. C.
Pass 7.500 369.1 .+-. 3.0 0.175 .+-. 0.042 F 2 5% DODAC + CpG
X-1857 Initial Pass 7.395 411.2 .+-. 5.0 0.153 .+-. 0.020 (20
.mu.g) + rH5 (10 .mu.g) X-1843 14 days @ 5.degree. C. Pass 7.38
394.7 .+-. 1.6 0.18 .+-. 0.036 F 3 % DODAC + X-18 8 Initial Pass
7.416 416. .+-. 5.3 0.173 .+-. 0.028 rH5 (10 .mu.g) X-18 8 3 days @
5.degree. C. Pass 7.492 395. .+-. 3.7 0.15 .+-. 0.013 TBD 14 days @
5.degree. C. -- -- -- -- F 5 5% DODAC + CpG X-18 0 Initial Pass
7.486 397.2 .+-. 6.9 0.175 .+-. 0.022 (10 .mu.g) rH5 (10 .mu.g)
X-1848 14 days @ 5.degree. C. Pass 7.553 3 7.1 .+-. 4.1 0.171 .+-.
0.028 F 7 5% DODAC + CpG X-18 2 Initial Pass 7.535 401.3 .+-. 1.5
0.168 .+-. 0.008 (5 .mu.g) rH5 (10 .mu.g) X-1848 14 days @
5.degree. C. Pass 7.731 400.4 .+-. 1. 0.158 .+-. 0.005 F 9 5% DODAC
+ CpG X-1864 Initial Pass 7.500 3 8.1 .+-. 7.7 0.177 .+-. 0.005 (10
.mu.g) rH5 (5 .mu.g) X-1850 14 days @ 5.degree. C. Pass 7.62 386.
.+-. 1.0 0.18 .+-. 0.013 indicates data missing or illegible when
filed
[0225] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described compositions and
methods of the invention will be apparent to those skilled in the
art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes
for carrying out the invention that are obvious to those skilled in
the relevant fields are intended to be within the scope of the
present invention.
* * * * *